You are on page 1of 127

DR MATHIAS WERNBOM (Orcid ID : 0000-0003-0411-3611)

Accepted Article
Article type : Review Article

Muscle fibre activation and fatigue with low-load blood


flow restricted resistance exercise – An integrative
physiology review
Mathias Wernbom1,2, Per Aagaard3
1
Center for Health and Performance, Department of Food and Nutrition and Sport Science,
University of Gothenburg, Gothenburg, Sweden. 2Department of Health and Rehabilitation,
Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg,
Gothenburg, Sweden. 3Department of Sports Sciences and Clinical Biomechanics, SDU
Muscle Research Cluster (SMRC), University of Southern Denmark, Odense M, Denmark.

Short title: Muscle fibre use in ischemic exercise

Corresponding author: Mathias Wernbom, Center for Health and Performance, Department
of Food and Nutrition and Sport Science, University of Gothenburg, Sweden. Box 300, SE-
405 30 Göteborg, Sweden. E-mail: mathias.wernbom@gu.se

Abstract

Blood flow restricted resistance exercise (BFRRE) has been shown to induce increases in

muscle size and strength, and continues to generate interest from both clinical and basic

research points of view. The low loads employed, typically 20-50% of the one repetition

maximum (1RM), make BFRRE an attractive training modality for individuals who may not

tolerate high musculoskeletal forces (e.g. selected clinical patient groups such as frail old

adults and patients recovering from sports injury) and/or for highly trained athletes who have

reached a plateau in muscle mass and strength.

This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/apha.13302
This article is protected by copyright. All rights reserved.
It has been proposed that achieving a high degree of muscle fibre recruitment is

important for inducing muscle hypertrophy with BFRRE, and the available evidence suggest
Accepted Article
that fatiguing low-load exercise during ischemic conditions can recruit both slow (type I) and

fast (type II) muscle fibres. Nevertheless, closer scrutiny reveals that type II fibre activation

in BFRRE has to date largely been inferred using indirect methods such as electromyography

(EMG) and magnetic resonance spectroscopy (MRS), while only rarely addressed using more

direct methods such as measurements of glycogen stores and phosphocreatine (PCr) levels in

muscle fibres. Hence, considerable uncertainity exists about the specific pattern of muscle

fiber activation during BFRRE.

Therefore, the purpose of this narrative review was (1) to summarize the

evidence on muscle fibre recruitment during BFRRE as revealed by various methods

employed for determining muscle fibre usage during exercise, and (2) to discuss reported

findings in light of the specific advantages and limitations associated with these methods.

Keywords: Occlusion, ischemia, muscle hypertrophy, motor units, fatigue

1. Introduction, background and purpose

2. Definitions and limitations

3. Neural control of muscle force development


3.1. Muscle fibre types and the size principle of motor unit recruitment
3.2. Can deviations from the size principle occur in voluntary movements?
3.3. Rate coding of motor units in voluntary movements
3.4. Can high threshold motor units fire at tetanic frequencies during maximal
voluntary contractions?
3.5. Type of activation versus firing rates for high threshold motor units
3.6. The possible roles of spike-frequency adaptation and small muscle afferents in
limiting maximal firing rates
3.7. The role of small muscle afferents in limiting muscle activation during fatiguing
exercise with large muscle mass

This article is protected by copyright. All rights reserved.


4. Fatigue and recovery in the muscle fibre types with ischemic exercise
4.1. Fatigue in the muscle fibre types during ischemic and anaerobic exercise
4.2. Possible contributions of low-frequency fatigue and neuromuscular
transmission
Accepted Article
failure to torque decreases in ischemic exercise
4.3. Force recovery in the muscle fibre types after ischemic and anaerobic exercise

5. Methods to investigate muscle fibre activation and use


5.1. Glycogen depletion
5.2. Phosphocreatine depletion
5.3. Electromyography (EMG)
5.3.1. Surface EMG
5.3.2. Intramuscular EMG
31
5.4. P-MRS
5.5. Studies on combined EMG and 31P-MRS

6. Studies on muscle fibre activation and use in BFRRE


6.1. Studies on glycogen depletion in BFRRE
6.2. Studies on phosphocreatine depletion to assess muscle fibre use in BFRRE
6.3. Summary of studies on glycogen and phosphocreatine depletion in BFRRE
6.4. EMG studies on BFRRE
6.4.1. Surface EMG
6.4.2. Intramuscular EMG
31
6.5. P-MRS studies on muscle fibre recruitment in BFRRE
6.6. Can the order of muscle fibre recruitment be changed during cuff occlusion?

7. Conclusions and implications


7.1. Order of muscle fibre recruitment in BFRRE
7.2. Patterns of muscle fibre use and fatigue in BFRRE
7.3. Consequences for hypertrophic adaptations in BFRRE
7.4. Consequences for neural adaptations to BFRRE
7.5. Consequences for interpretations of surface EMG during BFRRE
7.6. Consequences for the possible role of ”metabolic stress” in BFRRE

8. References
1. Introduction, background and purpose

Resistance exercise with low-to-moderate loads combined with blood flow restriction (BFR)

has been shown to induce increases in muscle size comparable to those seen with

conventional heavy resistance exercise in untrained individuals1-3, although a recent meta-

analysis4 suggests that the muscle strength gains are somewhat lower with BFR resistance

exercise (BFRRE). Nevertheless, the marked neuromuscular adaptations observed with

This article is protected by copyright. All rights reserved.


BFRRE continue to generate considerable interest from both clinical and basic research

points of view since the loads used in BFRRE typically are only ~20-50% of the 1 repetition
Accepted Article
maximum (1RM), to compare with the ~70-85% of 1RM that is often recommended and used

in conventional strength training (reviewed by Wernbom et al5 and Hughes et al6).

The stimuli for the muscle hypertrophy resulting from BFRRE, also known as

occlusion training or ischemic strength training, are not well understood at present. However,

it has been proposed that achieving a high degree of muscle fibre recruitment in BFRRE is

important1,5,7,8, and a number of studies have reported evidence suggesting that fatiguing low-

load exercise during ischemic conditions can recruit both slow (type I) and fast (type II)

muscle fibres (reviewed by Wernbom et al5, see also Suga et al9 and Takada et al10). The

physiological significance of type II fibre recruitment is that these fibres typically increase

their cross-sectional area to a greater extent than type I fibres with conventional heavy

strength training11-14, and that when measured at 37°C, human type II fibres have 3-fold

greater maximal contraction velocities and 4-fold higher maximal power outputs than type I

fibres15. Against this background, the considerable hypertrophy in both type I and type II

fibres reported by Nielsen et al16 after a short period of low-load high frequency BFRRE is of

great interest.

Taken together, it would appear that there is good evidence for the recruitment

of both fibre types with BFRRE. Nevertheless, closer scrutiny reveals that type II fibre

activation in BFRRE has to date largely been inferred via indirect methods such as

electromyography (EMG) (e.g., Moritani et al17; Takarada et al1,18; and Wernbom et al19) and

magnetic resonance spectroscopy (MRS) (e.g., Suga et al9 and Takada et al10), and only rarely

with more direct methods such as the measurement of glycogen and phosphocreatine (PCr)

levels in muscle fibres (e.g. Krustrup et al20; Cumming et al21). Moreover, in contrast to

This article is protected by copyright. All rights reserved.


Nielsen et al16, other studies have shown fibre hypertrophy and myonuclear addition

primarily in the type I myofibres with low-load BFRRE22-24.


Accepted Article
Therefore, the purpose of this review is to summarize the evidence on muscle

fibre recruitment in BFRRE as revealed by the various methods that have been employed for

determining muscle fibre use during exercise, and discuss reported findings in the light of

some of the advantages and limitations of these methods.

However, because motor unit recruitment, firing rates, substrate use and fatigue

all affect the physiological stimuli which the muscle fibres are exposed to, it was deemed

necessary to provide an introductory background of the physiology of these processes

(Sections 3 and 4) as well as a discussion of different methods to investigate motor unit

recruitment and muscle fibre use (Section 5). In section 6, the evidence of motor unit

recruitment and muscle fibre use in BFRRE will be outlined in detail. Finally, Section 7

discusses the possible influences of motor unit recruitment and firing rates on the resulting

neuromuscular adaptations to systematic training periods of BFRRE.

2. Definitions and limitations

For the purposes of this review, BFRRE is defined as exercise that is performed with typical

resistance exercise movements (e.g., knee extensions, leg presses, squats, elbow flexions)

with concurrent addition of BFR, most often provided by a tourniquet cuff around the

exercising limb although elastic wraps can also be used. In the current text low-to-moderate

load BFRRE will refer to a load range of ~20-50% of 1RM, unless otherwise indicated. In

some studies, the experimental resistance training protocols consisted of isometric muscle

contractions instead of dynamic resistance exercise. These exercise protocols were also

considered for this review. With isometric contractions, the intensity is typically expressed as

This article is protected by copyright. All rights reserved.


a percentage of the maximal voluntary isometric contraction (MVC) torque in the exercise,

and the load range referred to here is 20-50% of MVC.


Accepted Article
Because the most effective mode of blood-flow restricted training for increases

in strength and muscle size appears to be of the resistance exercise type (reviewed by

Wernbom et al5), the scope of this review is limited to BFRRE. With regard to the degree of

BFR, both very low levels of vascular restriction (e.g. Sumide et al25) and near-complete

occlusion (e.g. Shinohara et al26; Takada et al10) have been successfully employed to induce

measurable gains in muscle strength and mass. Therefore, studies covering this wide

physiological range are included and discussed in this review.

3. Neural control of muscle force development

The body of knowledge reviewed in this section is based on a vast number of studies that

have made motor unit recordings with needle or fine-wire electrodes, and additionally on

studies which have employed depletion of glycogen and PCr to investigate muscle fibre use.

Recent studies using sophisticated decomposition of surface electromyographic signals have

also been included as sources of information. See Section 5 for discussion on the some of the

advantages and disadvantages with these methods.

3.1. Muscle fibre types and the size principle of motor unit recruitment

It is generally accepted that with voluntary efforts, motor units are usually recruited in an

orderly manner based on the size of the motor neurons, so that small motor units comprised

of type I muscle fibres are recruited first, and with greater demands of force and/or velocity,

larger motor units with type II muscle fibres are increasingly recruited27-30. According to their

fatigue and contractile characteristics, motor units are usually classified into three major

This article is protected by copyright. All rights reserved.


types: slow fatigue-resistant (S) units, fast fatigue-resistant (FR) units, and fast fatigable (FF)

units31-33.
Accepted Article
However, this classification was originally based on studies on cat

gastrocnemius muscles, and attempts to reproduce this scheme in humans have resulted in a

more continuous distribution of contractile properties, without clustering into discrete groups

(reviewed by Heckman & Enoka30). It deserves to be noted, however, that in the paper where

Burke et al32 corroborated their proposed classification system, they also remarked on the

wide and continous range of twitch contraction times found within each class of units, noting

that while the distribution of fatigue resistance of fast units had a bimodal pattern (i.e., FR

and FF), it was essentially also continuous. They also found a few units that were

intermediate between FR and FF and which could not be classified as either type. This later

led to the identification of a fourth type of unit termed F(int), which is intermediate between

FR and FF, and which is sometimes also included in the proposed scheme of motor units34-36.

Furthermore, it is worth mentioning that the human studies which failed to

identify discrete groups of motor units (see Heckman & Enoka30) typically were performed

on small muscles in the hand and the foot. Burke36 suggested that in large limb muscles,

”distinct” types of motor units may be found while in smaller, distal muscles their

distributions are more continuous. In line with this notion, Garnett et al37 studied the human

medial gastrocnemius muscle using intramuscular electrical microstimulation and reported

that the motor units identified could be divided into S, FR and FF units. Nevertheless, the

distribution of contraction times of muscle fibre bundles varies even among relatively large

limb muscles in humans, with some muscles displaying a wide spectrum of contraction times

while others have less wide distributions38. The data reported by Buchtal & Schmalbruch38

suggest that the medial gastrocnemius muscle may indeed be one of the muscles which has

This article is protected by copyright. All rights reserved.


discernible gaps between the clusters of shorter, moderate and longer contraction times,

whereas many other muscles display a more continuous distribution.


Accepted Article
The motor units examined in intact human skeletal muscle correspond to the

three major fibre types that they typically consist of: type I, type IIA and type IIX; and in

addition the intermediate type IIAX which is a hybrid form containing both type IIA and IIX

myosin39-41. Thus, in a typical muscle, the type I fibres are activated first, followed by type

IIA and type IIAX and finally type IIX fibres42,43. With ATPase and immunohistochemical

methods, further hybrid types with varying extents of co-expression of type I and type IIA

myosin can be identified: type IC, type IIC and sometimes also type IIAC41,44. However,

these are normally rarely (~0.5-2%) seen in muscle biopsy samples even when all two or

three subtypes are considered together (see Staron41).

It should be noted that 100% pure type IIX fibres are also often rare, especially

in some types of athletes. As an extreme example, Andersen et al45, using electrophoretic

single fibre analysis, found only 0.1% pure type IIX fibres in the vastus lateralis muscles of

sprinters, in contrast to the 10.5-18.8% type IIX fibres they observed with traditional myosin

ATPase histochemistry methods. With gel electrophoresis analysis Andersen & Aagaard46

reported myosin heavy chain (MHC) IIX isoform content to be 9.3 ± 5.9% (±SD) in

untrained yet habitually physically active males. Using similar methods, Klitgaard et al47

found only 1% pure type IIX fibres in the biceps brachii of bodybuilders, as compared to

12% in control subjects, and that ATPase staining overestimated the percentage type IIX in

both groups. Ennion et al39 reported only 3% pure type IIX in the vastus lateralis of normal

healthy volunteers. It is thus clear that traditional staining methods result in an overestimation

of type IIX fibres, as most of the type IIX fibres identified by ATPase and

immunohistochemistry methods also express varying amounts of type IIA myosin41,45,47. In

recognition of this continuum, type IIAX fibres have sometimes been further subdivided into

This article is protected by copyright. All rights reserved.


IIAx, IIAX and IIXa depending on the balance between the type IIA and IIX

expression44.48,49.
Accepted Article
Interestingly, there appears to be a functional differentiation even among type I

myofibres, as indicated by the recent discovery of two metabolically distinct populations,

termed type I-1 and type I-2 fibres50. The former subtype has a smaller cross-sectional area

(CSA), a higher density of lipid droplets, and a tendency towards lower glycogen content

compared to type I-2 fibres. The subtype I-2 has similar lipid content and CSA to that of type

IIA fibres50. The implications of these two groups of type I fibres are not entirely clear, but

given the rather wide range of glycolytic capactity within the type I fibre population as

judged by the activities of key glycolytic enzymes51 and in vivo glycolytic rates52,53, we

speculate that the type I-2 fibres have a significantly greater glycolytic capacity than the type

I-1 fibres, perhaps somewhere in between that of type-I-1 fibres and type IIA fibres,

respectively. This would be in line with their intermediate position between these two fibre

types in the fibre type continuum as proposed by Prats et al50.

The size principle of motor unit recruitment was first demonstrated by

Henneman and colleagues in the 1950s and 1960s54,55, and essentially verified also in humans

in studies using motor unit recordings obtained with intramuscular electrodes56-61. Notably,

the size principle has also been found to operate in humans during both stretch reflex induced

contractions and with transcranial stimulation of the motor cortex62,63.

It has been generally held that motor unit recruitment in relatively large limb

muscles occurs up to ~85% of MVC, while in hand muscles recruitment is complete already

at ~50-60% of MVC27,28,64. However, accumulating evidence suggests recruitment of new

units up to as high as ~88-96 % of MVC in limb muscles such as tibialis anterior, soleus,

biceps brachii and vastus lateralis65-69. Nevertheless, Christie et al67, Oya et al68 and De Luca

& Hostage69 all employed slow ramp maximal contractions, which may well entail some

This article is protected by copyright. All rights reserved.


fatigue already during the contraction (see Section 3.5.), which in turn can raise the

recruitment threshold of the highest threshold units (discussed in Section 3.4.). As a result,
Accepted Article
the approximate upper limit for motor unit recruitment during more normal and

physiologically relevant contraction durations (e.g. 1-3 seconds) remains to be fully defined.

3.2. Can deviations from the size principle occur in voluntary movements?

Possible exceptions to the size principle have been suggested, mainly falling into the

categories of very rapid (ballistic) contractions, lengthening (eccentric) contractions and

muscle actions during changed afferent input such as cutaneous stimulation (reviewed by

Bawa et al70). Although some studies have reported deviations from the size principle during

ballistic and eccentric contractions71,72, subsequent studies by other researchers have for the

most part failed to confirm these findings (reviewed by Chalmers29, Heckman & Enoka30, and

Bawa et al70, but see also Grimby73 for some possible reasons behind different results).

Current evidence thus supports that the size principle operates in most types of human

voluntary movements, including ballistic and eccentric contractions.

However, an unequal balance of excitatory and inhibitory inputs onto motor

neurons may give rise to departures from the orderly recruitment74. Some of the more

dramatic changes in recruitment order in both animals and humans have been observed with

electrical stimulation of cutaneous afferents, resulting in elevated recruitment thresholds in

the lower-threshold units and vice versa for higher-threshold units61,75,76. Masakado et al76

observed that nearly all the units that normally were recruited above 30% of MVC had

lowered thresholds after cutaneous stimulation. It is important to note that the findings in

these studies do not fulfill criteria for a systematic overall reversal of the recruitment order77.

For example, cases of increased and unchanged thresholds among the high-threshold units

This article is protected by copyright. All rights reserved.


can also be found in the data of Masakado et al76, where the typical decrease among the high-

threshold (>30% of MVC) units seems to have been on the order of 5-10%.
Accepted Article
In other words, while some high-threshold units may become recruited earlier

than normal, a significant number of low-threshold units still appear to be recruited before

these high-threshold units. Furthermore, electrical stimulation of cutaneous afferents might be

considered a non-physiological condition compared with normal tactile stimulation78,79. Still,

the findings in the studies of Kanda et al75, Garnett & Stephens61 and Masakado et al76

constitute possible proof-of-principle that some deviations from the normal recruitment order

can occur when inputs from afferents are unequally distributed on the alpha-motorneuron

pool during voluntary contractions. Interestingly, increasing evidence suggests that such

alterations can also occur during painful contractions80,81. Even so, the physiological

relevance of these findings remains to be determined, as most of the human studies cited

above concerned contractions performed at only a few percent of the MVC, the study of

Masakado et al76 being a notable exception.

3.3. Rate coding of motor units in voluntary movements

The other major mechanism that the central nervous system (CNS) uses to regulate muscle

force development is “rate coding”, which refers to the modulation of the firing rates

(synonymous terms: discharge rates, firing frequencies) of the active motor units27,28. In most

muscles, force typically is increased by both motor unit recruitment and increased firing rates

up to ~85% of MVC, but above this level further increases in muscle force are considered to

be accomplished entirely by rate coding (reviewed by Duchateau et al28). A generalized

scheme of the size principle for the recruitment of motor units and the accompanying

increases in firing rates is depicted in Figure 1 below. Rate coding has a direct influence on

the force developed by the muscle fibres innervated by the firing motor neuron. In the human

This article is protected by copyright. All rights reserved.


quadriceps, the torque evoked by neuromuscular electrical stimulation (NMES) rises with

increasing stimulation frequency and reaches a plateau of maximum torque at ~50-60 Hz


Accepted Article
when the interval between pulses is constant82-84. Notably, studies on bundles of human

muscle fibres which were stimulated in vitro at 37°C demonstrated a similar force plateau at

~50-60 Hz85,86.

Interestingly, even at 100% of MVC, the average maximum firing rates

observed in human limb muscles during maximal contractions in vivo are typically only ~25-

30 Hz64,87, which is considerably lower than the 50-60 Hz required for maximum torque

development with NMES. As noted by Enoka & Fuglevand64, this suggests that either the

force is not maximal during these presumed maximal contractions or that the force exerted by

a motor unit does not depend solely on its average discharge rate. There may be several

explanations for the discrepancy between the maximal firing rates observed during MVC in

vivo and the stimulation frequencies required for maxinal isometric torque with NMES.

Firstly, the CNS may use very short intervals between the first firings, resulting

in double discharges (doublets) when a contraction is initiated, especially when the intent is

to perform the contraction as fast and strong as possible60,88,89. It is known that doublets and

triplets markedly enhances force when added to a submaximal firing rate via the so-called

catch-like property of skeletal muscle27,90,91. This force enhancement may last up to a few

seconds before it dissipates27,64. Such short bursts of extra impulses both in the beginning and

later on in the contraction89 may in part explain the paradox of how high forces can be

reached during maximal voluntary contractions despite seemingly suboptimal firing rates

when averaged across af few seconds.

This article is protected by copyright. All rights reserved.


However, it has been reported that average maximal firing rates in the vastus

lateralis can increase with strength training92,93, and that several measures of activation levels
Accepted Article
of the quadriceps correlate well with the measured maximal firing rates in the vastus

lateralis94. Moreover, although earlier studies reported near-maximal activation of the

quadriceps during MVC (e.g. Woods et al95), several more recent studies have found that

even with maximal voluntary effort, quadriceps muscle activation is typically only about 85-

90% during isometric and slow dynamic contractions96-101, although still others have found

near maximal (~95%) voluntary activation in the vastus lateralis (e.g. Pucci et al102).

The study of Pucci et al102 is of particular interest, as it demonstrated not only

high voluntary activation of the quadriceps but also relatively high maximal firing rates

during MVC (~42 Hz). Notably, the discharge rates increased drastically from ~20 Hz to ~42

Hz between 75% and 100% of MVC. This clearly indicates the importance of approaching

maximal voluntary activation in order to reach near-maximal firing rates even when these are

expressed as mean values for all units. Collectively, these findings on voluntary activation

and motor unit discharge rates strongly suggest that firing rates in the quadriceps during

MVCs are often not high enough to elicit the maximum torque capacity of this muscle in

subjects without resistance training experience.

Secondly, it is important to note that many of the investigations on firing rates

versus force development have reported average maximal rates. It is known that human

muscles contain motor units with a wide spectrum of contraction times of the muscle fibres

ranging from slow to fast38, which is probably explained by the continuum of fibre types and

subtypes discussed earlier in Section 3.1. In the study of Buchtal & Schmalbruch38, there

were 3-4 fold differences in contraction times between the fastest and the slowest bundles of

muscle fibres in the biceps brachii and triceps brachii when the bundles were activated in situ

in human subjects by intramuscular stimulation. In line with these estimates, Faulkner et al15

This article is protected by copyright. All rights reserved.


reported a 3-fold difference in maximal shortening velocity between bundles of human type

II fibres and bundles of type I fibres stimulated in vitro at 37° C.


Accepted Article
The short contraction times of the type II fibres means that these require much

higher discharge rates to reach tetanic tension than type I fibres68,103,104. This notion is

supported by the in vitro data in the report of Moulds et al86. Although 50-60 Hz was required

for generating maximal force in a given biopsy sample, 80% of maximum force was reached

already at ~15 Hz in bundles consisting of 80% type I fibres, whereas bundles made up of

57% type II fibres required ~30 Hz to reach 80% of maximal force86. In line with this,

Grimby et al103,105 reported that fast motor units with contraction times of 40 ms had maximal

firing rates of ~50 Hz (calculated from periods of 250 ms) in vivo, whereas slow units with

contraction times in the range of 60-90 ms had maximal firing rates of ~30 Hz. The fastest

units observed by Grimby, Hannerz and colleagues65,105 were characterised by maximal firing

rates of 65 Hz. Even greater ranges were found by Bellemare et al104, who reported that the

mean maximal firing rates for the biceps brachii and the adductor pollicis varied largely in

proportion to their respective twitch contraction and half relaxation times and that for each

muscle, the firing rates distribution covered approximately a fourfold range about the mean

value. Given the wide range of firing rates during maximal contractions, it can be argued that

reporting only average maximal firing rates provides relatively limited information on motor

unit behaviour during contractions of maximal efforts.

Thirdly, the time window used for calculating the discharge frequency has

varied considerably between studies; e.g. from the shortest interval of four102 and five

consecutive spikes67,94,106,107 to the greatest number of discharges in 250 ms intervals65,108 and

1 second intervals104,109. This can in part explain the discrepancies in the maximal firing rates

between different studies even when the same muscles have been investigated. For the biceps

brachii, Christie et al67 reported 37 Hz compared with 31 Hz in Bellemare et al104, whereas

This article is protected by copyright. All rights reserved.


Gydikov & Kosarov109 found no units firing higher than 24 Hz even at 100% of MVC.

However, Gydikov & Kosarov109 investigated firings rates only when steady-state forces had
Accepted Article
been reached, which probably also contributed to the low values. The importance of the

criteria for firing rate calculations is further underscored by the study of Van Cutsem et al89,

who observed that the interspike intervals increased for the first four discharges during

ballistic contractions, and that the increase was particularly marked for the interval between

the third and fourth spike. The increase in interspike intervals with increasing numbers of

successive discharges is likely at least in part due to spike-frequency adaptation, discussed in

Section 3.6.

Taken together, these observations help explain the discrepancy between the

maximal firing rates required for maxinal force development during electrical stimulation and

those observed during MVCs in vivo. Nevertheless, as will be outlined below, it may well be

that not all motor units tetanize during maximal effort contraction.

3.4. Can high threshold motor units fire at tetanic frequencies during maximal

voluntary contractions?

A current debate revolves around the questions whether high threshold units reach firing rates

sufficiently high for tetanic tension during voluntary maximal activation, and if the discharge

frequencies of these high threshold units really are higher than those of the earlier recruited

low-threshold units. In a series of investigations spanning over several decades, De Luca and

coworkers presented evidence and arguments which indicate that the firing rates of earlier

recruited motor units are greater than those of later recruited motor units during voluntary

isometric contractions even up to 100% of MVC69,110-113. Several other investigations have

arrived at similar results68,114,115. This observed property of higher firing rates in the earlier

This article is protected by copyright. All rights reserved.


versus the later recruited units all the way up to maximum force development has been

termed the ”onion skin” phenomenon111.


Accepted Article
However, as reviewed by Enoka & Duchateau116 and Piotrkiewicz & Türker117,

other researchers have reported results which support that high-threshold units can reach

higher firing rates than low-threshold units during near-maximal to maximal

contractions65,67,68,105,106,108,109,118–120. Christie et al67 observed that motor units recruited at

higher forces tended to demonstrate higher maximal discharge rates, although the relationship

was weak (r2 = 0.08).

For several reasons, these contrasting findings are not necessarily mutually

exclusive. The first one relates to the previous point on whether the motor unit firing rates

observed during MVCs in humans are truly maximal. As argued above, this often appears not

to be the case for the quadriceps in untrained subjects. Given that motor units with the highest

recruitment thresholds generally also have the fastest contraction times71,103,105, these must be

activated with the highest discharge rates to reach tetanic tension, as discussed in Section 3.3.

with reference to type I and type II fibres. There is some evidence from other muscles than

the quadriceps that this is possible, but typically only for short periods in untrained subjects.

In a series of studies on the tibialis anterior and on the extensor digitorum brevis

muscles, Grimby, Hannerz and co-workers65,71,103,105,121 found populations of very high-

threshold units that typically fired intermittently in bursts lasting 100-200 ms with short

intervals in between, and that these could continue during maximal voluntary efforts for only

brief periods before ceasing to fire. This typically happened after a few seconds when the

discharge rates of these units had dropped to 25-30 Hz. As noted in the previous section, the

highest frequencies observed for this type of units were in the range of ~50-65 Hz.

Interestingly, Hannerz65 also reported that the units recruited above 80% of maximum

isometric tension in the tibialis anterior were all of the intermittently discharging type.

This article is protected by copyright. All rights reserved.


Similarly, Gatev et al122 reported the recruitment of intermittently firing motor units above

80% of MVC in the flexor pollicis brevis. The intermittent ”phasic” firing patterns which
Accepted Article
seem characteristic of these units73 and their near-maximal thresholds could mean that these

units are easily missed and/or difficult to follow in experimental settings. Grimby &

Hannerz71 raised a similar point in noting that continuously firing long interval motor units

were more easily identified than intermittently firing short interval motor units (the latter

presumably representing very high threshold units).

A second reason relates to a point raised by Hannerz65, who noted that

measuring ”maximum frequency” during sustained contraction based on the highest

discharge in only 250 ms increases the maximum frequencies as compared with those

obtainable from a whole second, but it does so significantly only for motor units with high

recruitment thresholds. From this and the earlier discussion in Section 3.3., it is clear that the

criteria for calculating the maximum firing rates of different units can greatly impact the

results and conclusions derived hereupon.

3.5. Type of activation versus firing rates for high threshold motor units

A third aspect, related to those discussed above, concerns the manner in which the

contraction is performed, and the impact that this has on the firing rates of fast motor units.

Grimby et al103 illustrated this by following the fastest motor unit that they observed in one of

their subjects during varying types of isometric contractions, and showed that this unit

increased its frequency in proportion to the rate of force development (Figure 5 in Grimby et

al103). Based on the criteria of Harwood et al107 of at least 5 consecutive action potentials,

approximate firing rates for this unit can be calculated. With slowly increasing contraction,

the unit fired at a relatively steady rate of ~30 Hz after about 2 seconds. During maximal

sustained contraction, the unit fired at ~50 Hz for periods of 100-200 ms. With an

This article is protected by copyright. All rights reserved.


accelerating but submaximal contraction, the unit reached as high as ~100 Hz for a time span

of ~50 ms. Finally, during ballistic contractions, it discharged at ~100-140 Hz for periods of
Accepted Article
~67 ms.

In the same paper by Grimby et al103, the authors provided an example of the

different dischage behaviour between two opposite types of motor units, continuously firing

long interval motor units and intermittently firing short interval motor units, possibly

consisting of type I and type IIX fibres, respectively (discussed in Grimby & Hannerz71).

During the first second of a strong sustained contraction, the intermittently firing unit

discharged at 40 Hz, which dropped to 20 Hz during the next second of the contraction. In

contrast, the continuosly firing unit maintained 20 Hz during the first 2 seconds of the same

sustained contraction. With prolonged contraction, only the continuously firing unit

responded.

Compelling evidence for the existence of intermittently firing high threshold

units in humans was presented by Warmolts and Engel123,124, who performed open biopsy

electromyography in the biceps brachii and the quadriceps in patients with low-grade

neuropathy but with otherwise normal levels of muscle strength. The biopsies were taken

from the muscles after motor unit patterns had been recorded in the same areas during

different types of voluntary contractions. A few samples were almost entirely (96-100%)

composed of type I and type II fibres, respectively. When the pre-biopsy recordings were

analyzed, it was found that the almost pure type I areas had sustained discharges throughout

the entire force range, with maximum rates of 18-20 Hz at full voluntary efforts. Conversely,

the nearly pure type II areas were only active during strong or sudden activations, discharged

only for brief periods between 0.5 to 5 seconds, and reached peak frequencies of between 16-

50 Hz. Although caution is warranted due to reinnervation of denervated muscle fibres,

Grimby125 has argued that abnormal firing behaviour is seen only during the early phase of

This article is protected by copyright. All rights reserved.


reinnervation, and that at later time points the firing behaviour is no different from normal

motor units.
Accepted Article
Further support for the importance of the type of activation on motor unit

discharge rates comes from intramuscular electrode studies on dynamic movements, which

have shown markedly greater firing rates with concentric contractions compared with

isometric contractions, even when using the same relative intensity for each type of

contraction107,126. Harwood et al107 showed that low-load (25% of MVC) dynamic elbow

extensions with maximal effort resulted in greater firing rates in the anconeus muscle than

isometric contractions at 100% of MVC (39 Hz vs 24 Hz). Moreover, Grimby73 noted that

although lower-threshold units could also reach very high firing rates during ballistic

contractions, higher-threshold units reached the greatest discharge frequencies. Similarly, the

results of Desmedt & Godaux60 suggest higher firing rates in higher-threshold versus lower-

threshold units during ballistic contractions.

Collectively, this evidence clearly indicates that high-threshold units can reach

higher discharges rates than low-threshold units during brief maximal contractions,

particularly during accelerating, fast dynamic and ballistic contractions. This conclusion is in

line with Enoka & Duchateau116, who with reference to gradual increases in force stated that

”recordings of motor unit activity with fine-wire electrodes in which action potentials can be

observed directly often show that the peak discharge rate achieved during such tasks is

greater for later recruited motor units”.

It must again be stressed that the time periods during which the highest

threshold units reach their peak rates are typically very brief. Summarizing their results on

maximal voluntary contractions in the tibialis anterior and the extensor digitorum brevis

muscles, Grimby73 noted that ordinary subjects did not drive the highest thresholds units for

more than a few seconds, despite having done their utmost to the point of being very

This article is protected by copyright. All rights reserved.


exhausted. Possible causes of this inability in untrained subjects to maintain the firing in the

highest threshold motor units include motor unit adaptation (also known as spike-frequency
Accepted Article
adaptation, see below) and a selective increase in the recruitment thresholds for these units

due to fatigue (reviewed by Grimby73,127, see also Grimby et al105).

3.6. The possible roles of spike-frequency adaptation and small muscle afferents in

limiting maximal firing rates

With slow ramp contractions and maximal contractions lasting longer than a few seconds,

high-threshold motor units do not necessarily reach higher discharge rates than low-threshold

units. It deserves to be noted that the MVCs in the studies of De Luca and colleagues69,110-113

were performed with gradually increasing effort for about 10 seconds before the MVC was

reached, i.e. a rate of 10% MVC/s. During such conditions, the onion skin pattern of firing

rates of the recruited units may well prevail over other patterns. Interestingly, Erim et al112

noted that during such contractions, the subjects failed to reach their pretest MVCs, which

were performed during 3 seconds with no restrictions on the rate of torque increase. Erim et

al112 conceded that their subjects could have been affected by fatigue during their slow ramp

contractions. Inspection of their figures suggests that their subjects only reached ~90% of

MVC during the 10 s ramp contractions.

Since the same type of slow ramp contractions were used in many of the reports

from De Luca and coworkers, it is possible that the highest threshold units never reached

maximal firing rates in these studies. This possibility is supported by the findings of Oya et

al68, who studied motor unit recruitment in the soleus muscle during slow ramp contraction at

a rate of 10% MVC/s. They noted that while higher threshold units reached higher maximal

firing rates than lower-threshold units, the motor units recruited above 89% of MVC showed

peak discharge rates which were as low as those units recruited at or below 30% of MVC. A

This article is protected by copyright. All rights reserved.


similar tendency may be discerned in the results of Christie et al67, who also used slow-ramp

MVC contractions at a rate of 10% MVC/s. In Fig 2 in Christie et al67, the motor units
Accepted Article
recruited at or above ~87% of MVC seem to generally fire about ~10 Hz slower than the

units recruited between ~80-86%. The surface EMG data in the same study reveal a clear

trend towards a decreased mean power frequency (MPF) during the last 3 seconds of the 10-

second ramped-up MVC, suggesting development of fatigue.

Further evidence for the negative effects of previous muscle activity on the

maximal firing rates comes from a study by Van Cutsem and Duchateau128, who observed

decreased discharge rates in motor units during ballistic contractions which were preceded by

3-4 second isometric contractions at 25% of MVC compared to contractions initiated from a

relaxed state. Interestingly, the lower firing rates during ballistic contractions performed

immediately after low-intensity contractions were noted also in high-threshold motor units

that were not recruited at 25% of MVC, which suggests that the 25%-contraction negatively

affected the entire motor unit population. Harwood et al129 also reported lower firing rates in

maximal velocity contractions when these were preceded by submaximal fatiguing

contractions. Taken together, these findings strongly suggest that near-maximal firing rates

may not be reached in the highest-threshold motor units during slow isometric ramp

contractions, and that this type of contraction does not reflect the behaviour of these units

during the very activities that they are primarily designed for, i.e. brief very strong and fast

contractions.

The explanation may at least in part reside in the phenomenon of spike-

frequency adaptation in the motor units, which refers to the decrease in motor unit firing rate

as a function of time130. It has been shown that this acute phenomenon has an early rapid

phase which lasts ~2 seconds, which is then followed by a late phase of adaptation, with most

of the decline taking place during the initial phase130. One of the underlying mechanisms is

This article is protected by copyright. All rights reserved.


afterhyperpolarization, which is longer in slow motor units and shorter in fast units131-133. The

extent of spike-frequency adaptation appears to be greatest in the fast-fatigable units and least
Accepted Article
in the slow fatigue-resistant units130. This may in part explain both why very high firing rates

cannot be maintained during MVCs, and why maximal discharge rates are not seen in the

highest-threshold units during slow ramp contractions. This notion conforms well to the

observations that these units have a much higher fatigability also in humans37 and that type

IIXa and IIAx muscle fibres have the longest recovery times after PCr depletion due to

intense exercise49.

However, with training and extraordinary motivation, it is possible to drive

high-threshold units for longer than a few seconds at a time73. Given that the discharge rates

of intermittently firing units can decline already after the first second of high-force

contractions103, an improved ability to drive very high-threshold units seems likely to be one

of the major mechanisms among the host of so called neural adaptations, and the strength

increases that result from systematic training. It would also appear that if the adaptations are

especially pronounced in these units, they could easily be missed in studies on firing rates

before and after a period of strength training if only mean maximal values are reported. This

risk is underscored by the estimate that motor units innervating type IIX myofibres may

comprise only about 4% of the motor units while contributing with as much as ~18% of the

muscle fibres in a limb muscle such as triceps brachii64.

Another pathway which can impact on motor unit discharge rates originates

from small diameter afferents in the muscle. Classic physiological experiments have shown

that fatiguing contractions result in lower firing rates in the motor units, that complete

occlusion of the blood flow to the muscles after exercise maintains the suppression of firing

rates, and that these recover to normal values only once the circulation is restored95,134.

Bigland-Ritchie and colleagues134 suggested that group III and IV muscle afferents were

This article is protected by copyright. All rights reserved.


ideally suited to mediate such reflex modulations in motor unit firing rates during acute

fatigue and it is now well accepted that increased firing in these afferents leads to reduced
Accepted Article
motor neuron firing, The precise pathway for this effect is not known135, but it has been

proposed that group III/IV afferents act upstream of the motor cortex to inhibit voluntary

descending drive136.

How group III and IV afferents affect the different populations of motor units is

also not entirely clear, but it has been suggested that they decrease the firing rates of fast

fatiguing units in particular137, which in humans would be those with type IIX and IIAX

fibres. In contrast, others have provided arguments and evidence suggesting that group III/IV

afferents primarily affect low-threshold units, leading to slowing or derecruitment of these

small units80,81. However, the observation that saline injection and post-exercise ischemia

affect group III/IV afferent input differently (discussed in Martin et al.138) warrants caution in

extrapolating findings from saline injection studies to fatigue occuring during physiological

conditions. Interestingly, firing of group III and IV afferents may also influence

afterhyperpolarization of spinal motor neurones and thereby possibly also affect spike-

frequency adaptation, although the effects of this input during repetitive motor unit firing

remain to be elucidated (discussed in Windhorst et al137).

Spike-frequency adaptation may in part explain some paradoxical findings in

the literature on motor unit firing rates during fatigue. Garland et al139 observed that during an

isometric contraction of the triceps brachii at 20% of MVC held to fatigue, the motor units

which were active already from the beginning of the task (presumably S and perhaps also

some FR units) typically displayed marked decreases in their firing rates throughout the

contraction even as task failure neared, although by then some early recruited units had

started to increase their rates again. In contrast, motor units which were recruited later in the

contraction, which were identified as higher-threshold units, typically increased their firing

This article is protected by copyright. All rights reserved.


rates throughout the remainder of the task, with only a few exceptions. Figure 1 in Garland et

al139 also suggests that some of the lower-threshold units may have ceased to fire after
Accepted Article
reaching their lowest discharge rates, but this was not commented by the authors. It is

relevant to note that blood flow in the triceps brachii can become occluded already at 25% of

MVC and that sustained contractions at 20% of MVC are followed by pronounced

hyperemia140,141, suggesting that the muscle blood flow is insufficient at this load. Therefore,

the findings of Garland et al139 have possible implications for the activation of motor units

during ischemic exercise as well as for the interpretation of EMG results from BFRRE

studies, which will be discussed later (Section 7.5.).

3.7. The role of small muscle afferents in limiting muscle activation during fatiguing

exercise with large muscle mass

Studies have shown that skeletal muscle afferent input (including group III/IV afferents) to

the CNS increases with the amount of active muscle mass142-145. Interestingly, this muscle

mass dependence of afferent input to the CNS also affects endurance and muscle activation in

different tasks. Thus, Matkowski et al146 found greater losses in MVC torque and voluntary

activation (assessed by interpolated twitch analysis) after unilateral compared to bilateral

isometric knee extensions sustained to fatigue at 20% of unilateral and bilateral MVC,

respectively. Notably, despite similar ratings of perceived exertion (RPE) at the end of the

tasks (20 on the Borg 6-20 RPE scale), endurance time was shorter and EMG increases in the

quadriceps were reduced in the bilateral task. Resting doublet twitch force decreased after the

unilateral task only.

Similarly, Rossman et al147 demonstrated greater peripheral fatigue (e.g.

amplified decreases in MVC and twitch force) in unilateral dynamic knee extensions than in

bicycle exercise when both were performed to task failure. In another study, Rossman et al148

This article is protected by copyright. All rights reserved.


reported greater peripheral fatigue in unilateral than in bilateral dynamic knee extensions

performed to task failure, as judged by greater EMG increases and decreases in MVC and
Accepted Article
twitch forces. Rossman et al148 suggested that the CNS tolerates a greater magnitude of

peripheral fatigue when the source of group III/IV afferent feedback is limited to a small

muscle mass. Taken together, these findings are highly relevant for the activation of motor

units during BFR training with small and large muscle mass exercises, as discussed in greater

detail in Section 7.2.

4. Fatigue and recovery in the muscle fibre types with ischemic exercise

4.1. Fatigue in the muscle fibre types during ischemic and anaerobic exercise

A discussion on the fatigue processes in the muscle fibres during ischemic conditions is

motivated by the fact that fatigue is inherent and constitutes a major driving factor behind

muscle fibre recruitment in BFRRE (reviewed by Wernbom et al5). In an early study on

human muscle during ischemia, Buchtal & Schmalbruch38 showed that 20-45 minutes of

circulatory occlusion eliminated electrically induced twitch contractions of slowly

contracting muscle fibres in the biceps brachii, the triceps brachii and the gastrocnemius

muscles, while faster contracting fibres still responded to twitch stimulation with

characteristically short contraction times. In contrast, Dietz149 suggested that high threshold

motor units rather than low threshold units are inhibited during acute ischemia. Nevertheless,

studies on rat muscles have shown a greater decline in twitch force in slow muscle than in

fast muscle during the first 45 minutes of ischemia, particularly during the first 15-30

minutes150,151. Likewise, Gossen et al152 reported markedly greater declines in twitch force in

slow contracting motor units than in fast units during very low-force (~5-10% of MVC)

voluntary ischemic contractions in human subjects.

This article is protected by copyright. All rights reserved.


The available evidence thus suggests that during rest and very low-force

contractions, slow motor units are relatively more sensitive than fast motor units to ischemia
Accepted Article
of short to moderately long durations. This may be explained by the greater oxygen

consumption at rest in slow muscle compared with fast muscle153. Studying stimulated low-

force contractions in rat soleus muscle under anaerobic conditions, Sahlin et al154 concluded

that the decline in relative force was similar to that previously observed in fast twitch muscle

and that the soleus cannot be termed fatigue-resistant under anaerobic conditions. In should

be noted that with very low-force (~5-10% of MVC) voluntary ischemic contractions, it is

likely that only type I fibres are used155. With greater voluntary efforts due to higher loads

and/or fatigue in type I fibres, the type II fibres most likely will also be recruited and undergo

fatigue during ischemic exercise, and probably eventually to a greater degree than type I

fibres, as discussed elsewhere in this review.

The mechanisms of fatigue in muscle fibres during ischemic contractions are

probably in part related to the effect of hypoxia on PCr levels. Ischemia directly induces

muscle fatigue in humans, and the decrease in twitch force with increasing occlusion pressure

is proportional to the decrease in tissue oxygenation156. Intramuscular oxygen partial

pressures (pO2) have been correlated to the PCr levels during rest and exercise in humans157.

Bylund-Fellenius et al157 proposed that the correlation between the pO2 and PCr can be

explained by the equilibrium maintained by creatine kinase, since a direct relationship was

also found between the ATP/ADP ratio and PCr. During high energy demand, ATP is

initially relatively constant while PCr breaks down to creatine (Cr) and inorganic phosphate

(Pi)158. Increased Pi is one of the major causes of fatigue and can together with decreased

calcium (Ca2+) release from the sarcoplasmic reticulum (SR) explain much of the decline in

muscle force159.

This article is protected by copyright. All rights reserved.


Recent evidence also shows that increased hydrogen ions (i.e., lower pH) can

influence fatigue by acting synergistically with elevated Pi to depress force160,161 and that by
Accepted Article
considering these factors together with compromised Ca2+ release from the SR, it is possible

to account for much of the observed losses in force and velocity162. Strong support for a

synergistic role for pH on fatigue-induced force depression can be found in an in vivo study

on humans by Hultman et al163, who used neuromuscular electrical stimulation (NMES) to

evoke contractions during circulatory occlusion at a force corresponding to 50% of MVC,

with or without oral administration of ammonium chloride (NH4Cl) to vary intracellular pH

during exercise. Hultman et al163 showed that lower pH levels at the same degree of PCr

depletion were associated with a significantly lower force production after a 75-s ischemic

continuous isometric contraction, ending at 45% vs 55% respectively of the initial force level.

This investigation was one in a series of landmark studies in which Hultman

and colleagues investigated muscle fibre metabolism in the human quadriceps during

complete occlusion (~250 mm Hg) combined with NMES contractions52,163-169. These studies

helped to shed light particularly on the regulation of glyogenolysis and on the fatigue

processes during anaerobic exercise. Of particular interest for the physiology of BFR

exercise, Greenhaff et al52 reported more than 10-fold increases in glycogenolytic rates in

type I fibres in vastus lateralis muscles after NMES combined with occlusion compared with

fibres obtained after NMES alone. In contrast, the glycogenolytic rates in type II fibres with

occlusion were only marginally (not significant) higher than those observed during the free

blood flow condition. The NMES protocol included 20 maximal 1.6 s isometric contractions

with 1.6 s rest between each contraction, with a stimulation frequency of 50 Hz.

It can be argued that repeated 50 Hz stimulation is not physiological, but studies

from the same group in which 52 contractions with 20 Hz were combined with occlusion

resulted in even higher lactate levels in mixed muscle samples than in Greenhaff et al52 as

This article is protected by copyright. All rights reserved.


well as very low PCr values in both type I and type II fibres (Hultman & Sjöholm166,

Söderlund & Hultman167, Söderlund & Hultman169). The lactate in mixed muscle increased to
Accepted Article
128.9 mmol per dry weight, corresponding to ~29.6 mmol per kg wet weight, assuming a

77% water content in human skeletal muscle170. Such extreme lactate values in mixed muscle

fibres are only possible if glycogenolysis is extensive in both fibre types. Thus, ischemia can

drastically increase lactate production and decrease muscle fibre pH in type I fibres also

during muscle contractions at physiological firing rates.

Additional evidence for an important role of low pH in ischemic exercise

fatigue can be found in the investigation of Hultman & Sjöholm166. In their report, it was

shown that isometric force was still relatively well maintained (~80% of baseline) after 26

ischemic contractions, when PCr had already reached minimal levels. After 39 and especially

after 52 contractions, the force decline was much greater and mirrored the continued increase

in lactate levels, while PCr was essentially constant. However, it is possible that the low PCr

levels and consequent Pi increases may after a delay have resulted in Ca2+-Pi-precipitation in

the SR, causing decreased Ca2+ release from the SR158,159. In addition, the decreased pO2 may

reduce Ca2+ release within a few minutes as will be discussed below. Interestingly, the force

decline in the study of Hultman & Sjöholm167 was also related to decreases in ATP, which

dropped to as low as 37% of baseline levels, and to the decline in the ATP/ADP ratio. Very

low ATP levels, which can be especially pronounced in type IIX fibres following exhaustive

muscle exercise, may affect force and velocity negatively via multiple mechanisms, several

of which ultimately decrease the Ca2+ release from the SR158. Collectively, the drops in PCr

and ATP, and the corresponding changes in Pi, pH and ADP along with decreased Ca2+

release, probably explain a large part of the peripheral fatigue accumulated during ischemic

contractions.

This article is protected by copyright. All rights reserved.


Of note, Tesch et al171 and Söderlund & Hultman169 demonstrated 11-13%

higher resting PCr concentrations in type II than in type I fibres. This was confirmed and
Accepted Article
extended by Karatzaferi et al49, who showed that the resting PCr levels were highest in type

IIXa fibres (composed of 50-100% type IIX MHC) and lowest in type I fibres, with the type

IIXa fibres displaying ~33% higher values than type I. In contrast, type IIA fibres and type I

fibres had similar PCr levels, resulting in 11% greater PCr content in the type II fibres

overall. In the same study by Karatzaferi et al49, 25 seconds of maximal sprint cycle exercise

produced similar low absolute PCr levels the different fibre types immediately post-exercise,

with the type I fibres only slightly higher than the type II fibres. These findings strongly

suggest that the percentage drop in PCr levels can be greater in type II fibres with exhaustive

anaerobic and ischemic exercise and that the corresponding increase in Pi will also be greater

in type II fibres, particularly in the fastest subtypes. This may contribute to the greater torque

decreases seen with repeated concentric contractions in individuals with a high percentage of

type II fibres170, and to the greater fatigue in individuals with higher percentage type IIX

compared to persons with lower type IIX fibre proportion but who otherwise demonstrate the

same overall % of type II fibres172.

In addition to decreased PCr and pH levels, cellular ischemia can also suppress

Ca2+ release from the SR. Sun et al174 demonstrated that hypoxia (1% O2) markedly depresses

Ca2+ release and force development in comparison to normoxia (20% O2), with intermediate

forces at 5% O2. Interestingly, the effects of hypoxia were mediated by a decreased

production of hydrogen peroxide (H2O2) from NADPH oxidase 4 (Nox4). Nox4 was found to

produce H2O2 in direct proportion to the pO2, and the authors further reported that consequent

oxidation of a small set of cysteine thiols on the ryanodine receptor (RyR1) results in

increased RyR1 activity and Ca2+ release in isolated sarcoplasmic reticulum and in cultured

myofibres and enhanced contractility of intact muscle. They proposed that Nox4 thus acts as

This article is protected by copyright. All rights reserved.


an O2 sensor in skeletal muscle. The effects of pO2 on Ca2+ release and contractile force were

seen after only 10 minutes of hypoxia. Furthermore, a study on rat skeletal muscle showed
Accepted Article
that slow red muscle fibres have >2-fold greater expression of Nox4 and >2-fold higher

baseline production of H2O2 than fast white muscle fibres175. This raises the intriguing

possibility that greater Nox4 expression and baseline activity may contribute to the seemingly

greater pO2-sensitivity of force production in type I myofibers.

4.2. Possible contributions of low-frequency fatigue and neuromuscular

transmission failure to torque decreases in ischemic exercise

A persistant lowering in the ratio between stimulated forces at low stimulation frequencies

(typically 20 Hz) and those obtained at higher frequencies (50-100 Hz) may be observed after

the exercise-induced metabolite changes have reverted back to baseline levels. This is

denoted as low-frequency fatigue, reflecting an impairment in the electromechanical coupling

and consequent reductions in Ca2+ release176,177. In support of this phenomenon, Wernbom et

al178 reported a decreased 20/50 Hz ratio in the vastus medialis 3 minutes after an acute

fatiguing low-load BFRRE bout, when the partial BFR (~55% of complete occlusion

pressure) was still maintained. The 20/50 Hz ratio was reduced by 34% (Wernbom,

unpublished results).

Importantly, decreased myofibrillar Ca2+ sensitivity by factors such as increased

Pi and lowered pH can cause a similar low-frequency fatigue158. The acute changes reported

by Wernbom et al177 could therefore be due to decreased myofibrillar Ca2+ sensitivity and/or

decreased Ca2+ release. In support of the former alternative, 20/50 Hz ratio was no longer

significantly changed at 1 hour post-exercise. However, Sieljacks et al179 reported a reduced

20/100 Hz ratio in the quadriceps as a whole at 1 hour and 1 day, respectively, after a similar

acute BFRRE protocol as that employed by Wernbom et al178. Thus, there is evidence for

This article is protected by copyright. All rights reserved.


both acute and prolonged low-frequency fatigue with acute BFRRE performed with multiple

sets to failure.
Accepted Article
Wernbom et al178 further reported 61-62% reductions in MVC torque during the

first 2 minutes post-exercise, and no improvement between the 1 and 2 minute post-exercise

MVCs during maintained partial BFR. Part of the decline during post-BFRRE MVCs with

occlusion still in place may be explained by a reduced central excitatory drive. With a similar

protocol, we observed that EMG in the quadriceps during 1 minute post-BFRRE MVC with

maintained BFR was markedly lower compared with pre-exercise MVCs (~30% reduction,

Wernbom unpublished data 2007). Similarly, Yasuda et al180,181 reported reduced EMG

activity in the biceps brachii during post-exercise MVCs with sustained partial BFR, and

even greater decreases in EMG after exercise with complete occlusion. Motor unit

derecruitment and reduced firing rates would act in concert with an impaired

electromechanical coupling and decreased Ca2+ sensitivity, which in turn would contribute to

decrements in MVC torque.

However, the acutely reduced 20/50 Hz ratio seen after fatiguing BFRRE by

Wernbom et al178 could also in part be due to a decreased excitability of the muscle fibres

and/or transmission failure in some fibres. Importantly, both these factors may affect the

EMG signal amplitude165,182. Hultman & Sjöholm165 measured EMG in the quadriceps during

a continuous electrically stimulated (20 Hz) contraction which lasted 75 seconds, and showed

decreased EMG in parallell with decreased force, thus demonstrating that decrements in

EMG can have a peripheral origin. The contraction started at ~70% of MVC and ended at

~50% MVC. At 50-75% of MVC an almost complete occlusion of the blood flow occurs in

the quadriceps due to the high intramuscular pressures183,184, thus the EMG decreases in the

investigation of Hultman & Sjöholm165 were likely caused by the combined effects of

ischemia and the prolonged contraction. In line with this scenario, Cupido et al185 showed in

This article is protected by copyright. All rights reserved.


the human biceps brachii that continuous 20 Hz stimulation in combination with ischemia

(300 mm Hg) caused a decrease in the M-wave size after about 60 s, which continued to
Accepted Article
decrease steeply toward values approaching zero during the next ~60 s.

Working with the tibialis anterior and soleus muscles, Galea186 confirmed these

findings with 20 Hz and 30 Hz stimulation. In parallell with the decline in M-wave

amplitude, tetanic torque decreased, reaching 4-20% of control values after 3 minutes. In

contrast, little or no changes were seen at the same time points during 20 Hz stimulation with

free circulation185,186. Studying voluntary low-force contractions in the soleus, Leonard et

al187 found a 52% reduction in M-wave readings after 105-126 coupled concentric-eccentric

repetitions performed in approximately 3-4 minutes during ischemia (300 mm Hg). A

lowered M-wave size suggests a decreased effectiveness of neuromuscular transmission

and/or action potential propagation in muscle fibres188, respectively, although how M-wave

changes should be interpreted is debated189. Nevertheless, the consistent decreases in M-wave

size reported by Leonard et al186, Cupido et al184 and Galea185 as well as in the evoked EMG

responses by Hultman & Sjöholm165 are very similar to those seen with high-frequency

fatigue190. Thus, acute EMG and M-wave decreases of this magnitude seem to be associated

with a reduced muscle fibre excitability and/or neuromuscular junction (NMJ) transmission

failure.

With regard to the latter, Dahlbäck et al191 studied impulse transmission during

ischemic contractions in human extensor digitorum communis muscles. In the recordings of

action potentials from pairs and sometimes also three and four muscle fibres that belonged to

the same motor unit, they found that one of the fibres in a pair dropped out after a certain

number of discharges, and that a total block typically occurred after between 3500-7000

impulses. The block was ascribed to failure of neuromuscular transmission at the motor end-

plate. Notably, at a rate of ~10 Hz, a 50% block of one of the potentials occurred after 6.5

This article is protected by copyright. All rights reserved.


minutes, and in recordings of units with three and four fibres, the first fibres dropped out

already after 2.5-3 minutes, which corresponds to a total of about 1500-1800 muscle fibre
Accepted Article
action potentials. Another interesting finding was that when there were only 30 seconds of

rest with free circulation between the periods of ischemic contractions, the time until these

changes occurred was ~35% shorter. Dahlbäck et al191 emphasized that the synthesis of

acetylcholine is an aerobic process which is probably negatively affected by the lowered pO2

during ischemia. They concluded that during ischemia, the synthesis of acetylcholine is likely

inhibited and that the block occurs when synaptic stores of acetylcholine have been emptied.

Presynaptic failure of neuromuscular transmission can also occur in the motor

axon, especially at the branch points. Sieck & Prakash192 proposed that the probability of

axonal conduction block may be greater in FF units, due to the typically larger size and

greater number of axonal branches in these. They also suggested that this might explain the

observation of Sandercock et al193 of failure to evoke muscle fibre action potentials in some

of the muscle fibres in a given motor unit. The findings of Sandercock et al192 of blocked

action potentials mainly concerned the responses to a 80 Hz stimulation protocol which

induced high-frequency fatigue. Nevertheless, during high-frequency fatigue, there appears to

be more or less parallell decreases in M-wave, EMG and force (Figure 5 in Bigland-

Ritchie190). Notably, ischemia is known to slow motor nerve conduction velocity within 10-

15 minutes during resting conditions194,195. Given this and the striking similarities between

the effects of high-frequency fatigue and the fatigue induced by low-to-moderate load

ischemic contractions on these parameters, it seems possible that axonal conduction block

can occur in some branches during ischemic exercise.

Ultimately, neuromuscular transmission efficacy depends on the end-plate

potential (EPP), which in turn can be impaired by both pre-synaptic and post-synaptic

mechanisms192. Muscle fibre excitability is an important post-synaptic factor, and the

This article is protected by copyright. All rights reserved.


combination of lowered acetylcholine release and decreased fibre excitability can result in

EPP rundown and eventually transmission failure196. It seems clear that ischemic exercise can
Accepted Article
negatively affect both pre and post-synaptic mechanism, which may explain the findings of

Dahlbäck et al191. Moreover, results from rat muscle studies show that FF and F(Int) units are

generally more prone than FR and S units to M-wave decreases during repetitive

stimulation192. Studies on rat muscles also indicate that fast fibres have a much greater Na+-

K+ leak/pump ratio than slow fibres, and the resulting faster rise in extracellular K+ and

decreased Na+ gradient results in an accelerated loss of excitability in fast fibres197. Even so,

it seems reasonable to suggest that repetitive stimulation during ischemia can lead to

transmission failure also in some type I and type IIA muscle fibres, given that the S and FR

units are likely recruited much more extensively than F(Int) and FF units during low-force

contractions. This notion is supported by the fact that the motor units investigated by

Dahlbäck et al191 were primarily very low-threshold ones (see also Section 6.6).

The observation that some fibres, probably including type I fibres, can ”drop

out” due to neuromuscular transmission failure already after 2.5-3 minutes of relatively

moderate firing (~10 Hz) during ischemia has implications for muscle fatigue also during

low-load BFRRE. Although these studies investigated muscle contractions during complete

vascular occlusion, their findings suggest the possibility that the decreases in EMG in post-

BFRRE MVCs observed by Yasuda et al180,181 and in our labs (Wernbom et al. unpublished

results 2007) could in part have been due to transmission failure in some of the muscle fibres,

in addition to varying degrees of reduced excitability in active muscle fibres and lower firing

rates in many of the motor units.

Regardless of the relative contributions from each of the above fatigue

mechanisms, it is clear that there is a substantial peripheral fatigue component in BFRRE

which does not fully recover until free blood flow is restored. This notion is in line with

This article is protected by copyright. All rights reserved.


studies showing that if blood flow occlusion is maintained or applied immediately after

fatiguing exercise, there is no recovery of static and dynamic torque134,172. The dynamic peak
Accepted Article
torque results of Colliander et al172 even suggest a ~10% further decrease after a 1 minute rest

period with complete occlusion. This observation sheds light on the seemingly small

magnitude of post-exercise recovery of MVC reported by Yasuda et al181 and Wernbom et

al178 while partial BFR was still maintained. Fatigue will undoubtedly affect the force

development at the muscle fibre level, and hence also influence motor unit recruitment

patterns as well as modulate the mechanical and metabolic stimuli involved in BFRRE, as

discussed in detail below.

4.3. Force recovery in the muscle fibre types after ischemic and anaerobic exercise

In addition to depletion, Söderlund & Hultman169 also studied the recovery of PCr and ATP

levels in type I and type II fibres at different time points after ischemic contractions.

Although PCr levels were extremely low in both fibre types immediately post-exercise, a

faster repletion of PCr occurred in type I fibres than in type II fibres after 60 s recovery (to

~59% of baseline vs ~37%) with intact circulation. After 5 minutes of rest, PCr was no longer

significantly lower than baseline, although visual inspection of their graphs suggests that for

type II fibres, PCr was still ~22% lower while the type I fibres were back to pre-exercise

levels. Interestingly though, at 15 minutes post exercise, type II fibres had PCr levels which

were ~18% higher than baseline. The ATP levels were reduced to 60% and 53% in type I and

II fibres respectively immediately post-exercise, increasing to ~76-81% and ~61-63% at 20-

60 s post-exercise. At 5 minutes, the corresponding values were ~87 for type I fibres, and

~73% for type II fibres. At 15 minutes, ATP was no longer significantly lower than baseline

in type I fibres (~94% of pre) while type II fibres had still not recovered (~77%). Curiously,

these values are slightly lower than previously reported by Söderlund & Hultman167 using an

This article is protected by copyright. All rights reserved.


identical exercise protocol, where ATP levels were also reduced at 15 minutes post-exercise

in the type II fibres to ~91% of pre-exercise levels.


Accepted Article
These results suggest that full recovery of PCr levels in type II fibres after

severely depleting exercise may take considerable time, on the order of 10 minutes.

Furthermore, recovery of ATP after depleting ischemic exercise seems to take even longer

time in the fast fibres (>15 minutes). Unfortunately, no analysis of type II fibre subtypes was

conducted in the study by Söderlund & Hultman169. However, yet other studies have shown

that the rate of PCr recovery differs between the fibre types and subtypes after strenuous

anaerobic exercise, being quicker in both type I and pure type IIA fibres than in hybrid type

IIAX and almost pure IIX fibres49. After 1.5 minutes of recovery, PCr in type I fibres were

~64% of baseline levels, while type IIA levels were ~57%. In contrast, PCr levels in type

IIAx and IIXa (15-50% and 50-100% type IIX MHC, respectively) had only recovered to

~37% and ~33%. The rate of recovery of PCr after strenuous high-repetition isokinetic

exercise was positively correlated with the content of the oxidative enzyme citrase synthase

(CS), a marker of myocellular oxidative capacity198. Furthermore, type IIA fibres have

markedly higher oxidative capacity compared with type IIX fibres51,199-202. Taken together,

these results show that type I fibres replenish their PCr levels faster than type II fibres, and

that type IIA in turn recover considerably faster than both the hybrid type IIAX fibres and

pure IIX fibres.

Nevertheless, the significance of exercise-induced depletions and subsequent

recovery in PCr and ATP levels for acute muscle force capacity is not entirely clear. Sahlin &

Ren203 investigated the time courses of recovery for both MVC and various muscle

metabolites (including ATP, ADP, PCr and lactate) after sustained isometric knee extensions

at 66% of MVC which were held to fatigue (to ~50% of MVC). They showed that although

PCr was markedly depleted immediately post-exercise in mixed muscle samples and that

This article is protected by copyright. All rights reserved.


neither PCr nor lactate recovered to pre-exercise levels even with 4 minutes of rest (~85% of

pre-exercise for PCr), MVC was no longer significantly depressed at 2 minutes post-exercise
Accepted Article
(~95% of pre-exercise) and was fully recovered at 4 minutes of rest. Notably, MVC had

recovered to 80% already after 15 seconds of rest and to 90% after 30 seconds. Data from a

study with a very similar exercise protocol204 suggests that at these time points, PCr recovers

to only ~30% and ~40% of the baseline levels. Altogether, these observations confirm that of

Hultman & Sjöholm166 that even quite large degrees of PCr depletion are by themselves not

necessarily sufficient for large decrements in torque development, and therefore that there has

to be other factors behind the recovery of muscle force capacity in addition to PCr repletion.

In their study on repeated high-repetition isokinetic exercise, Jansson et al198

found that peak torque recovery between the sets was positively related not only to PCr

content relative to the resting values (r=0.63) but also to the degree of restoration of the

ATP/ADP ratio (r=0.69). Importantly, both these parameters as well as lactate recovery

correlated with CS activity, which in turn correlated with torque recovery (r=0.69). Using a

similar isokinetic protocol, Colliander et al172 showed that individuals with a dominance of

slow fibres (57% relative area of type I fibres) had an almost complete recovery of torque

after already 60 seconds of rest. In contrast, a group with a type II relative area of 70% had

only recovered to 80% of pre-exercise torque. As both groups had a mix of fast and slow

fibres, the differences in the in situ force recovery between type I and type II fibres were

likely even greater. Therefore, it seems clear that short rest intervals have a much more

negative effect on the recovery of force development in type II fibres than in type I fibres,

and that the recovery is probably more prolonged in the fastest subtypes of type II fibres (IIX,

IIAX) compared with type IIA.

This article is protected by copyright. All rights reserved.


With regard to BFRRE, it is important to note that the rest periods between sets

are not only typically brief (30-60 s), but also that most models of BFRRE involve occlusion
Accepted Article
also during the inter-set rest periods, which means that torque recovery between sets is

limited (see further below). However, it is likely that the force recovery in type I fibres is still

faster than in type II fibres. It should also not be overlooked that some studies have reported

signs of muscle damage and prolonged torque decrements lasting up to 48 hours post-

exercise after low-load BFRRE178,179,205 accompanied by low-frequency fatigue for 24 hours

post-exercise179. Cumming et al21 reported heat shock protein (HSP) responses from the same

acute BFRRE investigation as Wernbom et al178, and found that the torque decrements at 24

hours post-exercise were highly correlated with the cytoskeletal levels of HSP27 (r=0.87).

These findings may have implications for mechanisms of muscle damage, torque decrements

and low-frequency fatigue with BFRRE. A suggested scheme for some of the possible factors

behind the development of fatigue in BFRRE is depicted in Figure 2.

5. Methods to investigate muscle fibre activation and use

5.1. Glycogen depletion

Glycogen depletion has a central place in the history of the study of motor unit recruitment

and muscle fibre use. In the 1960s and 1970s, measurements of glycogen deposits in muscle

fibre sections provided direct evidence for the existence of different types of motor units in

both animals31,32,206 and humans37. Glycogen depletion was also used in many of the classic

studies on muscle fibre recruitment in various types of exercise in humans154,207-212 and

continues to be used up to the present day to map muscle fibre use with various exercise

protocols (e.g. Cumming et al21, Andersen & Gruschy-Knudsen213). In exercise studies,

glycogen content is usually determined semi-quantitatively with assessments of periodic acid

This article is protected by copyright. All rights reserved.


schiff (PAS) staining intensity on muscle fibre sections. Importantly, PAS staining intensity

on sections correlates very strongly (r=0.91-0.93) with direct measurements of glycogen


Accepted Article
concentration in the same muscle fibres210,214.

In contrast to endurance and intermittent exercise, relatively few studies had

examined glycogen depletion and repletion patterns in muscle fibres with resistance type

exercise up until the late 1990s215,216. Surprisingly, this is still largely true, especially

regarding the influence of resistance exercise variables on glycogen utilization and repletion

in the different muscle fibre types. Among the few studies on this subject, Robergs et al214

and Tesch et al216 demonstrated that glycogenolytic rates in mixed muscle fibres and in fast

fibres increased with increasing exercise intensity. This is supported by data from Söderlund

et al168, who showed that 50 Hz stimulation of the vastus lateralis resulted in a nearly doubled

rate of glycogenolysis in type II fibres compared with 20 Hz, which is consistent with the 50-

60 Hz required for maximal force in type II fibres. Current models of the regulation of

glycolysis during muscle contractions strongly suggest that both increased Ca2+

concentrations and elevated levels of ADP and AMP play important roles in activating

glycolysis217. More marked increases in Ca2+, ADP and AMP would thus appear to explain

the greater glycogenolytic rates in high-force contractions compared with low-force

contractions.

Robergs et al214 studied the effects of two resistance exercise protocols with

knee extensions at 70% and 35% of 1RM respectively, which were approximately matched

for total work (6 sets of 6 repetitions vs. 6 sets of 12-13 repetitions). Interestingly, although

the rate of glycogenolysis was greater for the higher load, the two protocols resulted in

similar degrees of glycogen depletion in both type I and type II fibres. Additionally, the data

suggested that glycogen utilization tended to be greater in the type II fibres, especially for the

high-load regime. Also of note, the rate of glycogen resynthesis during the first 2 hours post-

This article is protected by copyright. All rights reserved.


exercise was high particularly in the type II fibres. The higher rate of resynthesis in fast fibres

may in part have been due to glycogen synthesis from lactate218-220, which is considerably
Accepted Article
faster in type II fibres than type I fibres at least in rodents221 and which appears to take place

via a reversal of pyruvate kinase action218,219. As will be discussed later, an enhanced

glycogen repletion may also have influenced the results of studies on glycogen depletion with

BFRRE. However, Robergs et al214 did not differentiate between subtypes of type II fibres.

Tesch et al216 investigated three different resistance exercise protocols, each

consisting of 5 sets of 10 concentric-only repetitions for the knee extensors at 30%, 45% and

60% of 1RM respectively. Because they found very few type IIX fibres in their myosin

ATPase stained muscle sections, Tesch et al216 analyzed type IIAX and IIX fibres together.

With the two lower loads, only type I and type IIA fibres displayed reductions in glycogen,

but with 60% of 1RM, type IIAX+IIX fibres also showed glycogen loss. Interestingly,

Robergs et al214 reported no significant difference in type II fibre depletion between 70% and

35% of 1RM, and inspection of the absorbance data indeed suggests little difference: a ~52%

vs a 44% decrease in muscle glycogen levels in type II fibres with 70% of 1RM and 35% of

1RM, respectively. This suggests that type II fibres were recruited and used to almost the

same extent with the lower load protocol. In contrast, Tesch et al216 showed considerably

greater depletion in type IIA as well as type IIAX+IIX fibres with 60% of 1RM compared

with both 30% and 45% of 1RM.

Both Robergs et al214 and Tesch et al216 used subjects with resistance-training

experience, so a difference in training status seems unlikely to be an important factor behind

the divergent results. However, a greater number of repetitions was performed for the low-

load protocol in Robergs et al214 compared to Tesch et al216, ~77 vs 50 repetitions. An

important additional difference was that the repetitions were continuous concentric-eccentric

in the former study and concentric-only in the latter. As noted by Robergs et al214, an elevated

This article is protected by copyright. All rights reserved.


intramuscular pressure therefore remained throughout the duration of each repetition,

resulting in reduced blood flow, whereas the protocol of Tesch et al216 almost certainly
Accepted Article
allowed a higher muscle blood flow due to the muscle relaxation between each concentric

repetition (see Wernbom et al5 for further discussion of this point).

Taken together, the results of Tesch et al216 and Robergs et al214 demonstrate

that there is substantial type II fibre usage already at 30-35% of 1RM. However, the data in

Robergs et al214 suggest a relatively greater use of type II fibres with the low-load protocol in

their study, probably at least in part due to the maintained intramuscular restriction of blood

flow and resulting fatigue in type I fibres, which in turn may have caused compensatory

recruitment of more type II fibres, as discussed elsewhere in this review.

Robergs et al214 also showed linear decreases in glycogen levels in both type I

and type II fibres which were proportional to the total work performed during exercise. This

may be viewed as both a strength and a weakness. On one hand, a given pattern of glycogen

depletion may provide a rough estimate of how much a specific fibre type has been used in a

given exercise protocol. An important advantage is that full recovery of glycogen occurs

slowly (hours), thus biopsies do not have to be obtained immediately (within seconds) post-

exercise. On the other hand, because depletion is dependent on duration, it may require

several minutes before a measureable decrease is seen222,223, and as discussed elsewhere in

this review, the rate of glycogen depletion is usually much slower in type I than in type II

fibres with intermittent high-force contractions.

For example, Andersen & Gruschy-Knudsen213 found a much greater

percentage of glycogen depleted type IIX fibres compared to type I fibres after 340 maximal

concentric contractions (~70% vs 15%). Gollnick et al155 reported that repeated contractions

of the quadriceps at 20% of MVC or more sustained to fatigue resulted in glycogen depletion

of type II but not type I fibres. Similarly, Tupling et al224 reported non-significant decreases

This article is protected by copyright. All rights reserved.


in glycogen in type I fibres but substantial depletion in type II fibres with 5 second

intermittent contractions at 60% of MVC. The finding of seemingly very little type I fibre use
Accepted Article
at intensities 20% of MVC in the studies of Gollnick et al155 and Tupling et al224 is difficult

to explain but given that the contractions were performed many times and over periods of

many minutes, a considerable contribution from oxidative metabolism to the later bouts of

exercise is plausible, as observed by Ingemann-Hansen et al225. In contrast to these results

from repeated contraction studies, Tesch & Karlsson226 observed substantial and almost equal

lactate contents in type I and type II fibres after single contractions at 25%, 50% and 75% of

MVC maintained to fatigue. These and other investigations show that glycogen depletion

patterns need careful interpretation within the specific context that they occur.

Furthermore, it is known that muscle fibre type distribution can vary

significantly between the superficial and the deep parts as well as between the origin and the

insertion in human limb muscles such as the vastus lateralis and the biceps brachii227-229.

Interestingly, muscle fibre areas have also been reported to differ between the superficial and

deep layers in the vastus lateralis, with the mean fibre area being larger in the deep layers230.

It has been suggested that this is an indication of functional differences between the various

parts of the muscle230 (c.f. Section 5.3.1. for further discussion of such possible differences).

Accordingly, the possibility of differences in fibre activation patterns between different layers

in the same muscle must be kept in mind when choosing the sites of biopsy sampling and

when interpreting glycogen depletion patterns induced by various exercise protocols.

5.2. Phosphocreatine depletion

Pioneering studies on PCr depletion in human muscle fibres after acute exercise bouts were

published in the late 1960s and early 1970s231-233. Rehunen et al234 were the first to

investigate exercise-induced PCr changes in type I and II fibres separately, and seven years

This article is protected by copyright. All rights reserved.


later Tesch et al171 were the first to report on fibre-type specific changes in PCr with high-

force contractions. Tesch et al171 found reductions in PCr in type I and type II fibres to 41%
Accepted Article
and 31% of baseline levels immediately after 30 maximal concentric contractions at 180°/s,

thus providing clear evidence of both type I and type II fibre recruitment with isokinetic

resistance exercise. After 60 seconds of recovery, the levels were 69% and 49% of baseline

levels, indicating not only less decline in type I fibres but also a quicker recovery. It is

important to note that the concentric-only contraction were separated by 1.2 second rest

periods in the investigation of Tesch et al171, unlike with conventional resistance exercise,

where the concentric and eccentric phases follow each other with little or no relaxation in

between.

Since the study of Tesch et al171, surprisingly few investigations have looked at

PCr use in type I and II fibres during voluntary high-force contractions. A sensitive method

for determining muscle fibre use was developed by Beltman et al222, based on the ratio

between PCr and Cr levels. In a subsequent study, Beltman et al223 found that type I fibres

and some type IIA fibres had been recruited after 7 brief isometric contractions at 39% of

MVC, with further involvement of both type I and type IIA fibres after 7 contractions at 72%

of MVC. Only at 87% of MVC did type IIAX fibres display significantly lower PCr/Cr ratios

(type IIAX included fibres with 15-100% type IIX MHC). Employing this method, Beltman

et al99 also investigated muscle fibre use with maximal concentric and eccentric contractions

at 60°/s, as well as maximal isometric contractions, each performed for 10 repetitions. In this

study, the type IIAX included all fibres with 25-100% of type IIX MHC. Interestingly, while

all fibre types investigated showed decreased PCr/Cr ratios with all contraction types, the

shift was less in type IIAX fibres after eccentric contractions, indicating less recruitment of

these fibres during maximal eccentric exercise.

This article is protected by copyright. All rights reserved.


The paucity of research on fibre type use in resistance exercise as measured by

PCr depletion may in part be explained by the experimental difficulties associated with
Accepted Article
obtaining muscle biopsies quickly enough to ensure valid assessments of immediately post-

exercise PCr levels. For example, Beltman et al223 excluded muscle samples which had been

obtained ≥10 s post-exercise, which should be compared to the estimated half-time recovery

of 30 s for PCr levels. However, as indicated from the previous discussion on fibre type

differences in PCr recovery times, this half-time is almost certainly different between the

various fibre types and subtypes, with an approximate order from the longest to the shortest:

type IIX > type IIAX > type IIA > type I. Even so, the narrow time frame available for

muscle biopies for post-exercise PCr assessments means that the use of PCr depletion is

restricted to investigators who have developed great skill and specific routines for obtaining

muscle biopsies as quickly as possible (few seconds) following cesseation of exercise.

Furthermore, based on the finding that muscle PCr levels in muscle samples can

decrease with 13-19% with 1-4 weeks of storage even when the samples are stored at -70°C,

it has been recommended that PCr levels should be analyzed within 24 hours after the biopsy

sampling235. While it is well recognized that a delay between sampling and freezing of the

biopsy can affect muscle PCr values, the decreases with storage appear to be less known, and

many papers on PCr changes with exercise have not reported the time elapsed between

freezing and subsequent analyses of PCr levels. Both the delay between sampling and

freezing and between freezing and biochemical analyses could conceivably have affected the

results on resting PCr levels as well as changes resulting from exercise and recovery in many

of the papers in the literature reporting on these changes. Finally, it deserves to be noted that

measurements of PCr levels from muscle biopsy samples are subject to essentially the same

interpretive caveats as glycogen measurements with reference to possible differences in the

activation patterns between different regions of the same muscle.

This article is protected by copyright. All rights reserved.


5.3. Electromyography (EMG)

5.3.1. Surface EMG


Accepted Article
In exercise physiology, the type of EMG used is usually surface EMG with bipolar electrodes

applied on the skin over the working muscles. In surface EMG research, the amplitude is

often used to infer the level of ”neural drive” to the muscles, although the limitations of

surface EMG for this purpose have been known for several decades (reviewed by Duchateau

et al28, Farina et al236, and Vigotsky et al237). For example, Moritani et al238 cautioned that the

change in the surface EMG should not automatically be attributed to changes in either motor

unit recruitment or in firing rates as the EMG amplitude is further influenced by the

individual muscle fibre potential, degree of motor unit synchronization, and fatigue.

With the exception of sophisticated decomposition techniques235, it is probably

not possible to separate the contributions of recruitment of new motor units and increased

firing rates to increases in surface EMG amplitude during exercise, especially with dynamic

contractions. Even with advanced decomposition techniques, it can be very difficult to extract

neural strategies, as surface EMG provide only a limited representation of the active muscle

fibres and is biased towards the motor units with the largest surface action potentials, which

are large and superficial motor units236. Furthermore, surface EMG decomposition is possibly

sensitive to overlapping action potentials and phase cancellation117,240, although this has been

disputed241. In addition, decomposition techniques are still limited to mostly isometric

contractions and can not detect deep motor units236. For further discussion on these issues, the

reader is referred to the aforementioned papers, and to Farina et al242,243 and De Luca et al244.

A particularly serious criticism against using surface EMG to infer acute

changes in neural drive was raised by Dimitrova, Dimitrov and colleagues, who performed

simulation studies which suggested that peripheral changes in the muscle fibres themselves

due to fatigue may influence EMG amplitude to a much greater extent than changes in

This article is protected by copyright. All rights reserved.


”neural drive” 245,246. Specifically, lengthening of the intracellular action potential (IAP)

profile and the after-potential can cause increases in integrated EMG and EMG root mean
Accepted Article
square (RMS) amplitudes without alterations in neural input and recruitment of additional

motor units245,247. However, due to the lack of human data, Dimitrova & colleagues245-247

based their simulations of the effects of changes in the IAP profile primarily on data derived

from a study by Hanson & Person248 on frog muscle fibres investigated at room temperature.

Interestingly, Hanson249 noted that the after-potential in rat muscles was

considerably smaller than in the frog muscles studied earlier and that while the effects of

repetitive stimulation of rat extensor carpi radialis muscle on IAP parameters were of the

same kind as those in frog muscles, they were less pronounced. In rat soleus muscle fibres,

there were only very small changes249. In a subsequent study on both human and rat muscle

fibres, Hanson250 concluded that ”the intracellularly recorded potentials in human muscles

were not markedly changed during repetitive stimulation, while in the rat intercostal muscles

the same type of change was seen as earlier observed in fast limb muscle of rat and in frog

muscle fibres. Thus the human muscle fibres resemble, in this respect as well, the slow soleus

muscle of the rat”.

Because human muscle fibres apparently show much less fatigue-induced

changes in IAP than both frog and rat muscle fibres (except perhaps for rat soleus fibres), it is

possible that the relative degree of lengthening of the IAP profile was exagerrated in the

simulation studies of Dimitrova and colleagues245-247. Thus, the surface EMG amplitude

recorded from human skeletal muscle during fatiguing exercise may not be as affected by

changes in IAP profile as suggested in these simulation studies245-246, although the degree of

influence under hypoxic and ischemic conditions awaits further studies. In contrast, other

simulation studies suggest that amplitude cancellation of single motor unit action potentials

(MUAPs) can impact negatively on surface EMG amplitude, an effect which may be more

This article is protected by copyright. All rights reserved.


marked during fatiguing contractions251, although a later simulation study from the same

group reported negligible effects of fatigue on cancellation252. Surface EMG could thus still
Accepted Article
provide a useful, if crude, global measure of muscle activation, especially if EMG is properly

normalized30,253,254. However, recent results strongly indicate that the size of the MUAPs is

even more important than ”neural drive” for summated EMG amplitude and that normalized

EMG amplitude analysis may not be appropriate for inferring differences in ”neural drive”

between muscles255.

Nevertheless, EMG recording may with these limitations in mind provide clues

regarding possible differences in activation patterns in a single muscle. For example, Christie

et al67 showed weak correlations between motor unit firing rates and surface EMG RMS

amplitude over the entire range of isometric force development in the human biceps brachii.

Instead, they suggested that the overall trend of a slow initial increase followed by more rapid

increases in RMS amplitude at higher forces fitted with the later recruitment of superficial

motor units. A similar explanation was proposed by Moritani & Muro256, who also studied

the biceps brachii and who observed disproportionate increases in EMG RMS amplitude over

force output in the higher part of the force range investigated (0-80% of MVC). This scheme

is consistent with findings of Clamann257, who reported that motor units deep in the biceps

muscle had the lowest recruitment thresholds, and that those closest to the surface were the

last to be recruited. Combined, the results from these investigations on EMG in the human

biceps brachii strongly suggest that late recruited large superficial units may greatly

contribute to the overall surface EMG amplitude both by virtue of their size and by being

located nearer to the recording electrodes.

The deep-to-superficial recruitment pattern may also apply to the quadriceps.

Accordingly, it has been established that superficial layers in the vastus lateralis contain both

larger motor units258 and a greater percentage of type II muscle fibres229 than deeper layers,

This article is protected by copyright. All rights reserved.


and results from other studies are also consistent with a generally greater prevalence of type

II fibres in the superficial layers compared with the deeper layers in this muscle227,228,259. The
Accepted Article
vastus medialis muscle appears to have an even greater difference in fibre type distributions

between superficial and deep layers, with type II fibre dominance in the former and type I

dominance in the latter227. Furthermore, it has been reported that the vastus lateralis has a

greater percentage of type II fibres than the vastus intermedius259 and that while the rectus

femoris has a type II fibre dominance in all its parts, the superficial part has the greatest

percentage of type II fibres227. Interestingly, data from scanning studies indicate that the

vastus intermedius is the primarily recruited muscle in dynamic knee extension exercise at

low loads260-262, with all quadriceps muscle components being recruited at higher

loads260,261,263. Situated deep to the vastus lateralis and the rectus femoris, the vastus

intermedius is not accessible to surface EMG except at the most distal part264.

Collectively these findings suggest that a deep-to-superficial activation pattern

may to some extent exist not only within but also between muscle bellies in the quadriceps.

Thus, it is not surprising that numerous studies have demonstrated a non-linear relationship

between isometric knee extension force and surface EMG amplitude for the quadriceps

muscle264-269 although this is not always the case, see Alkner et al268 for discussion. In the

studies which have shown a non-linear relationship, the EMG-force curve typically seems to

be marked by a somewhat flatter slope at lower force levels accompanied by a steeper slope

at high to maximum force levels (see e.g. Thorstensson et al265, Rabita et al269, Watanabe &

Akima264).

Based on the findings of large superficial motor units in the vastus lateralis258

and that recruitment in this muscle may occur up to 95% of MVC69, it seems reasonable to

suggest that the relatively steep increase in EMG RMS activity seen between high and

maximal force development in the quadriceps may at least in part be caused by the

This article is protected by copyright. All rights reserved.


recruitment of very high-threshold superficial motor units containing type II fibres, possibly

type IIX. However, as discussed previously, Pucci et al102 showed that the discharge
Accepted Article
frequency in the vastus lateralis increased steeply from ~20 Hz to ~42 Hz when the torque

increased from 75% to 100% of MVC. Therefore, we suggest that higher firing rates may

also contribute to the steep increase in EMG RMS often seen between high and maximal

force development in the human quadriceps. Other factors such as motor unit

synchronization, amplitude cancellation and lengthening of the intracellular action potential

profile are also likely to affect the EMG amplitude, but their relative importance remains to

be determined.

An often overlooked phenomenon in conventional surface EMG is that EMG

amplitude during concentric contractions can exceed that observed in isometric

contractions270-273, especially with maximal efforts at fast contraction velocities270 and in the

initial phases of concentric contractions during conventional heavy resistance exercise272,273.

In the studies of Amiridis et al270 and Beutler et al273, EMG RMS activity recorded during

concentric knee extensor contractions temporarily reached up to ~1.6-1.8 times higher levels

than during isometric MVCs. It is important to note that in both these studies, EMG was

normalized to a 1 second period during the MVC which was either centered around the

greatest activation or after the force had reached a steady plateau. In contrast, the peak

dynamic EMG values were measured during time periods on the order of a few tenths of a

second. It is therefore likely that spike-frequency adaptation and consequent reductions in

maximal firing rates (Section 3.6.) affected the sEMG amplitude negatively to a much greater

extent in the MVCs than in the fast and accelerating concentric contractions in the studies of

Amiridis et al270 and Beutler et al273.

Initial spike-frequency adaptation, together with shorter time windows for

analysis of the EMG signal, probably also at least in part explains the greater EMG

This article is protected by copyright. All rights reserved.


amplitudes during fast vs slow concentric contractions which have been reported in several

studies270,274,275. Intriguingly though, EMG in the quadriceps can increase with ~20-30%
Accepted Article
during sets of repeated maximal-effort high-velocity pure concentric repetitions, peaking

after about 8-12 consecutive concentric contractions276. Given that high-velocity concentric

knee extensions already typically display the greatest EMG values274,275, it is highly unlikely

that this increase reflects improved muscle activation during the set. In this context, it is

interesting that changes in M-wave amplitude have been correlated with changes in EMG102.

Therefore, changes in muscle fibre excitability may at least in part explain the results of

Tesch et al276. In addition, it has been suggested that acute increases in M-wave and EMG

amplitudes may be misleading since intracellular muscle fibre action potential and muscle

fibre excitability can decrease at the same time189,236.

Taken together, it is apparent that there are many pitfalls in interpreting EMG

amplitude results247. Accordingly, it is suggested that possible changes in motor unit firing

rates and muscle fibre excitability (including transmission failure) must be taken into account

when interpreting EMG results from acute experiments and training interventions.

Furthermore, as surface EMG recording may not detect deep motor units236, there is a

theoretical possibility that some motor units might undergo drastic decreases in discharge

frequencies and/or in muscle fibre excitability and that some fibres may even drop out during

ongoing exercise without these events being detected.

5.3.2. Intramuscular EMG

Intramuscular electrodes are preferable over surface EMG to study the behaviour of single

motor units28, and many of the classic studies on motor unit recruitment have employed

intramuscular electrodes primarily in the forms of concentric needles and different variants of

fine wire electrodes28,277. Intramuscular electrodes have also been of central importance in the

This article is protected by copyright. All rights reserved.


efforts to determine the functions of the various muscles in the human body, as documented

in classic textbooks278. Intramuscular EMG can help distinguish between low-threshold and
Accepted Article
high-theshold motor units based on recruitment order and spike amplitudes17,279 and is

probably less affected by intracellular changes in muscle fibres than surface EMG245.

However, intramuscular electrodes do not necessarily reveal with certainty the

types of muscle fibres that are active. Furthermore, it is very difficult to use intramuscular

electrodes with dynamic movements, as they easily move out of position73. Needle and fine-

wire electrode EMG also have the obvious disadvantage of being invasive methods, and can

typically only track a few motor units at any given time, hence raising concerns about general

representivity. For example, the motor unit action potential amplitude detected with

concentric needle electrodes depends on muscle fibres located within a radius of 0.5 mm

from the active surface of the electrode280. Unless several electrodes are used (e.g. Moritani et

al279), whether the motor units recordings are representative must therefore be questioned.

5.4. 31P-MRS

Phosphorus-31 magnetic resonance spectroscopy (31P-MRS) is a non-invasive method which

has been used to study muscle metabolism in humans since the early 1980s281. Direct

measurements of pH and PCr in human muscle biopsies have confirmed the validity of 31P-

MRS for measuring exercise-induced changes in pH and PCr282, although other biopsy

studies have shown that decreases in pH and ATP may be somewhat overestimated with 31P-

MRS283. 31P-MRS has also been used for assessing muscle fibre recruitment. The peak of Pi

shifts during exercise according to changes in muscle pH, and the occurrence of ”split” Pi

peaks in the 31P spectra during exercise has been used to infer that both type I and type II

fibres have been recruited284. Specifically, the low pH peak of Pi is presumably caused by the

This article is protected by copyright. All rights reserved.


lower pH of type II fibres during exercise due to the greater dependence on glycolysis in

these fibres, whereas the high pH peak primarily reflects type I fibres284--287.
Accepted Article
Indeed, muscle biopsy studies have revealed that type II fibres have markedly

higher glycolytic rates during exercise than type I fibres in humans52,53,288. Furthermore,

several studies have shown a much faster disappearance of the high pH Pi peak vs the low pH

Pi during recovery after fatiguing exercise, which has been interpreted as reflecting the much

higher oxidative capacity of type I fibres compared with type II fibres, especially type IIX284-
286,289
. This interpretation is consistent with the observations that PCr recovery is dependent

on the magnitude of oxygen supply204,290,291, and that the higher the oxidative capacity of the

muscle fibre, the faster the recovery of PCr after exercise198.

Interestingly, studies have reported up to three Pi peaks in fatiguing exercise

during free flow conditions284,289, but in four out of seven subjects in the study of

Vandenborne et al284, only two peaks were observed. In Mizuno et al289, three Pi peaks were

observed only in 2 out of 14 subjects. The two peaks reported by Vandenborne et al284 were

interpreted as slow oxidative fibres (i.e. type I) and fast oxidative glycolytic fibres (type IIA)

respectively in some subjects, and fast oxidative glycolytic fibres and fast glycolytic fibres

(type IIX) in others. The recovery of the intermediate pH Pi peak was also much faster than

the low pH peak. This is in agreement with the higher oxidative capacity of the type IIA

fibres compared with type IIX fibres51,199-202, and also with biopsy studies on PCr content and

recovery after exhaustive sprint cycle exercise (discussed in Section 4.7.), where PCr

recovery is quicker in both type I and pure type IIA fibres than in hybrid type IIAX and

almost pure IIX fibres49.

Even so, criticism was raised already in early studies292-294 against data from

investigations where the signal had not been properly localized to one muscle belly.

Subsequent studies with localized 31P-MRS provided evidence for the split Pi being produced

This article is protected by copyright. All rights reserved.


by the different fibre types within the same muscle as opposed to pH differences between

different muscle bellies284,295. Experiments with relatively selective neuromuscular blockade


Accepted Article
of type I and type II fibres during exercise provided additional strong support for fibre type

heterogenity as the explanation for the Pi splits296,297. In these studies, blockade of either fast

fibres or slow fibres during exercise made the Pi split disappear compared to control exercise

without blockade296,297. Nevertheless, the use of surface coils larger than 2.5–2.9 cm on the

forearm is likely to sample signals from more than one muscle293. Because Mizuno et

al289,296,297 used a 4-cm coil and measured signals from multiple muscles, these elegant

studies are not definitive proof against the view that split Pi peaks can originate from different

muscle bellies, as opposed to (or in addition to) reflecting the recruitment of different muscle

fibre types in a single muscle. This notion is underscored by the results from T2 weighted

MRI studies on the plantar flexors, which show non-uniform increases in activity among the

individual muscles with exercise, demonstrating the gastrocnemius medialis to be the most

active agonist in plantar flexion exercise with the knee in an extended position298,299.

Indeed, two recent studies which employed precise localization methods to the

single muscle belly of gastrocnemius medialis demonstrated split Pi with non-localized 31P-

MRS but not with localized 31P-MRS300,301, which again has raised the question whether the

occurrence of split Pi peaks in single muscles is a fact or an artifact. Even among the studies

which have reported split Pi peaks, the occurrence has been variable. Vandenborne et al295

used localized 31P-MRS on the gastrocnemius medialis and lateralis and the soleus, and

reported that with steady state low frequency exercise, 5 out of 18 investigated muscles

showed a broad peak indicating 2 or 3 components, and in 2 of these muscles the broad Pi

peak clearly resolved into two distinct Pi peaks. With maximal effort high-frequency

exercise, the Pi line width was wider than the PCr width in all muscles, indicating more than

one component (and thus fibre type) in the Pi peak, but no split peaks were observed. This is

This article is protected by copyright. All rights reserved.


in clear contrast to their earlier study with the same maximal protocol but with a less precise

localization technique (87-90% of the signal from one muscle), in which 7 out 7 subjects
Accepted Article
displayed split Pi peaks284. Collectively, these results clearly indicate that signal

contamination from less active muscles may play a significant role in the observation of split

Pi peaks.

As an additional factor, the pattern of fibre type distribution also appears to play

a significant role in whether split Pi peaks are observed or not. Thus, Mizuno et al289 reported

that only subjects with a relatively even distribution of type I and type II muscle fibres

displayed split Pi peaks during exercise, whereas subjects with a predominance of type I or

type II fibres showed a single peak, at high and low pH respectively. Although this finding

should be viewed with caution due to the non-localized spectra, it suggests that a relative

abundance of both type I and II fibres is necessary to cause the ”compartments” in the spectra

that form the basis for split Pi peaks. Support for this possibility may also be found in the

results of a study by Houtman et al287, who observed a pH gradient during sustained isometric

contraction between the medal and lateral parts of tibialis anterior, and who reported double

Pi peaks in all their subjects during 30% of MVC sustained to fatigue. In addition, the failure

of some studies to find split Pi peaks with localized spectra of one muscle may be due to the

exercise not being strenuous enough to result in the recruitment of fibre populations

sufficiently different from each to cause Pi splitting.

A probably more consistent indication of recruitment of both type I and II fibres

during exercise is increased line width of the Pi peak, which is based on the same muscle

fibre pH heterogenity as split Pi peaks. Rossiter et al302 found that while splitting into low and

high pH peaks occurred in 5 out 11 subjects during exercise, all of them showed increases in

the Pi peak line width. Rossiter et al302 further noted that ”simple splitting of the Pi peak into

two separate, distinct regions may be an oversimplification. Although the Pi peak may be

This article is protected by copyright. All rights reserved.


adequately modeled by either one or two compartments, a broadening of the Pi peak in all

cases may reflect an overlapping of a number of muscle “compartments” distinguished by a


Accepted Article
range of pH values”. The difficulties with finding split Pi peaks in a single muscle are not

surprising in light of the evidence that in many muscles, there is a continuum of subtypes

within type I and type II muscle fibres, as discussed previously. Again, it should be noted that

the difference between type II and type I fibres in glycolytic rates and in accumulated lactate

levels can become substantially smaller during certain types of anaerobic exercise including

ischemic contractions.

This possibility is underscored by the findings of Tesch & Karlsson226,303, who

reported nearly as high lactate levels in type I fibres as in type II fibres in untrained subjects

after sustained contractions at 50% of MVC to exhaustion (up to 19.8-28.7 mmoles per kg

wet weight vs. 22.1-29.8 mmoles per kg ww). As previously noted, 50% of MVC is sufficient

to cause near-complete intramuscular occlusion of the blood flow in the human

quadriceps183,184. An increase to 22 mmol lactate per kg ww corresponds to a drop in pH of

about 0.5282,290. Interestingly, ~23 mmol lactate per kg ww has been reported in muscle fibres

after low-load BFRRE performed to failure225. Therefore, a significant shift in the Pi peak to

lower pH values may occur not only in type II fibres but in type I fibres as well during very

strenuous exercise, which could introduce methodological difficulties in separating the Pi

peaks. In line with this scenario, a late increased rate of deline in the pH of the high pH Pi

peak has been observed in the tibialis anterior muscle during ankle dorsiflexion isometric

contractions at 30% of MVC sustained to fatigue287,304.

Intriguingly, Houtman et al304 reported three phases of PCr usage during their

fatigue protocols. The first phase was characterized by a rapid decline in PCr, which was then

followed by a second phase of slowed PCr consumption, presumably due to increased

glycolysis and oxidative metabolism. During this phase, two distinct Pi peaks appeared which

This article is protected by copyright. All rights reserved.


were attributed to the contributions of type I and type II motor units. During the third and

final phase, a steep rate of decline in the pH of the high pH Pi peak was seen, and a merging
Accepted Article
of the two Pi peaks occurred in several subjects. Figure 4 in Houtman et al304 suggests that in

other subjects, the high pH Pi peak disappeared without any markedly increased rate of

decline in pH. This might be explained by trapping of Pi in mitochondria during ischemic

exercise, resulting in loss of the Pi signal (discussed in Houtman et al287). Notably, splitting

followed by merging of the two Pi peaks as well as disappearance of the high Pi peak have

been observed also during fatiguing dynamic exercise305.

In summary, it appears that split Pi peaks may reflect recruitment of both type I

and type II muscle fibres. However, the presence of inconsistent findings and the many

factors which can affect the occurrence and disappearance of the Pi peaks raise questions as

to whether determination of Pi splitting by 31P-MRS is sufficient as a stand-alone method for

assessing relative degrees of recruitment of the various fibre types.

5.5. Studies on combined EMG and 31P-MRS

MRS in conjunction with EMG is a useful approach in broadening the understanding of the

mechanisms of fatigue in health and disease306. A number of investigations have combinded

EMG and 31P-MRS to study the relationships between the myoelectrical and the biochemical

processes which underpin neuromuscular fatigue and recovery289,307-310. The studies of

Vestergaard-Poulsen et al308,309 are of particular interest with regard to muscle fibre

recruitment during fatiguing low-force exercise. In these studies, the subjects performed

isometric ankle dorsiflexion contractions at 10% and 30% of MVC to fatigue, and EMG

variables and metabolites in the tibialis anterior were monitored simultaneously. In both

studies308,309, it was reported that EMG RMS amplitude remained relatively constant or

increased only slightly during the low-force contractions until the pH value dropped below

This article is protected by copyright. All rights reserved.


6.8 and the PCr/ Pi ratio was between 1.0–2.0. Below these breakpoints, the EMG RMS

amplitude increased and in the latter study, a split Pi peak appeared synchronized with the
Accepted Article
increase in EMG. With further drops in pH and the PCr/ Pi ratio, EMG increased rapidly in a

hyperbolic manner to the point of exhaustion.

The findings of Vestergaard-Poulsen et al308,309 are very similar to those of

Houtman et al304, who studied tibialis anterior with an almost identical exercise protocol. In

Houtman et al304, the split Pi peaks appeared at around a pH of 6.8 in most of the subjects.

Houtman et al311 followed up their MRS study with an EMG study using the same exercise

protocol. An essentially hyperbolic EMG RMS increase was seen in several of their subjects

as they neared task failure, along with an increase and then a decrease in muscle fibre

conduction velocity. This was interpreted as recruitment of fresh type II muscle fibres due to

fatigue in type I fibres, with the type II fibres then showing fatigue toward the end of the

exercise. Importantly, this interpretation was supported by several lines of evidence.

These studies demonstrate some of the values of combining EMG and 31P-MRS

to gain better insight into the mechanisms of fatigue and the patterns of recruitment of motor

units during strenuous exercise. However, combining these two technologies is not without

problems as each of them might add unwanted noise to the recordings with the other312.

6. Studies on muscle fibre activation and use in BFRRE

6.1. Studies on glycogen depletion in BFRRE

At least two studies have shown type II fibre use in low-load BFR training via analyses of

glycogen depletion in muscle fibres from biopsies21,225. The study of Ingemann-Hansen et

al225 is arguably the first investigation of muscle fibre use in low-load BFRRE measured at

the cellular level. The exercise model was knee extensions performed in a prone position,

This article is protected by copyright. All rights reserved.


with intermittent working sets separated by 10 minute rest periods with free blood flow.

Muscle biopises were taken from the vastus lateralis. The combination of cuff width (7 cm)
Accepted Article
and pressure (300 mm Hg) used by Ingemann-Hansen et al225 typically results in complete

occlusion of thigh blood flow in most subjects in a supine position (Wernbom, unpublished

observations, see also Crenshaw et al313).

Unfortunately, these authors reported the load in Watts (14.7) and kilograms

(10), and not in % of MVC or 1RM. Based on the cadence (20 repetitions per minute) and

endurance time, the number of repetitions completed by their subjects in the first set of

exercise was around 55. From this and from the absolute load used (10 kg), as well as data

from our own acute experiments on dynamic knee extensions during varying degrees of

BFR19,178,314, it can be estimated that the load in the study of Ingemann-Hansen et al225 was

on the order of ~15-20% of 1RM. The subjects performed 10-16 sets of exercise until

exhaustion, and biopsies were taken immediately after set 2, 5, 8 and the last set (while the

cuff was still inflated), and 30 minutes after the last set.

The glycogen depletion data from this study indicated recruitment of both type I

and type II fibres, although the picture was complex. After the last exercise set, the glycogen

content was reduced by 55% and 39% in the type I and type II fibres, respectively.

Interestingly, the reduction was not homogenous in the type II fibres, with some fibres

showing only minor reductions (~10%) while others displayed more marked depletion

(~50%) and still others were in between these figures. Strikingly, the most depleted type II

fibres were those that had the lowest oxidative capacity, as judged by low staining for

NADH-tetrazolium reductase (NADH-TR). Conversely, the type II fibres with the highest

NADH-TR staining also had the highest glycogen content, i.e. they seemed to have been used

the least. This is a puzzling finding, given that the type II fibres which have the highest

oxidative capacity and NADH-TR staining are typically type IIA fibres and vice versa for the

This article is protected by copyright. All rights reserved.


type IIX fibres (type IIB in older literature)199-201,315. In other words, the fibres which were

presumably type IIX seemed to have been used to a much greater extent than those which
Accepted Article
presumably were type IIA. At a first glance, this could suggest an altered order of motor unit

recruitment (i.e., type I > type IIX > type IIA).

In order to make sense of these findings, a brief discussion of subtypes of type

II fibres is warranted. As noted above, type IIA generally have a greater oxidative capacity

than type IIX. Type IIAX, the intermediate form between type IIA and IIX, appears to be

close to IIA in oxidative capacity as judged by mitochondrial content and succinate

dehydrogenase activity200,315, but may have been interpreted as type IIX (previously called

IIB in humans) in older ATPase-based classification systems due to its intermediate to dark

myofibrillar ATPase stain at pH 4.6 (for discussion and pictures, see Billeter et al317, Staron

et al200; Sant’Ana Pereira et al40,48 and Staron44). And as discussed in Section 4.1., some of

the fibres which are classified as type IIX fibres by ATPase histochemistry often contain

significant amounts of type IIA myosin in addition to type IIX, resulting in an

underestimation of hybrid IIAX fibres and an overestimation of type IIX fibres.

Taken together, these findings may in part explain why some researchers did

not find a perfect correspondence between type IIA and type IIX staining on one hand and

NADH-TR (or lack of thereof) staining on the other199. In addition, performing histochemical

staining of myosin ATPase only at a single pH of 10.3-10.4 as done in Ingemann-Hansen et

al225 does not allow for separation between the subtypes of type II fibres, including the

relatively rare and higly oxidative type IIC and type IIAC fibres in addition to type IIA, IIAX

and IIX41,44. Nevertheless, type II fibres which stain clearly positive for NADH-TR are for

the most part type IIA fibres, while fibres that are only lightly stained are type

IIX199,202,315,316, with IIAX hybrids probably being closer to IIA than more pure IIX fibres on

NADH-TR stained sections because of their higher mitochondrial content.

This article is protected by copyright. All rights reserved.


Based on the above considerations we can now attempt to interpret the puzzling

findings of Ingemann-Hansen et al225. As judged by the glycogen depletion, it appears that


Accepted Article
the type I, type IIA and IIAX/IIX muscle fibres were all recruited by the BFRRE performed

in their study. But due to lower oxidative capacities in type IIX, these fibres were more

dependent on anaerobic glyogenolysis than the type IIA and perhaps also type IIAX fibres.

The type IIA fibres may have used oxidative phosphorylation of both glycogen and lipids to a

significant extent, since the lipid stores are about twice as large as in type IIX fibres200.

Interestingly, Ingemann-Hansen et al225 suggested that aerobic metabolism played a greater

role during the last bouts than during the first ones, and that this could have been facilitated

by greater oxygen stores, caused by a greater number of open capillaries. Such oxygen stores

would be used to a certain extent even though the blood flow was probably nearly completely

occluded during each bout.

The finding of greater glycogen depletion in both type IIX and type I fibres than

in type IIA fibres is not without precedent. Fridén et al320 reported that repeated 25 second

sprints on a treadmill caused glycogen depletion in type I and type IIX fibres, but not in type

IIA fibres in sprinters. It is of note that sprinting, like ischemic exercise, can drastically

increase the rate of glycogenolysis in type I fibres52,53. Assuming that the size principle holds

for both these modes of exercise, it is still difficult to explain why the presumably heavily

recruited type IIA fibres should show less glycogen depletion than both type I and IIX fibres.

However, it is known that type IIA fibres have a lower tension cost than type IIAX fibres321,

and thus probably also compared with type IIX. As noted earlier, type II fibres have been

shown to replete glycogen faster than type I fibres214,322. In an investigation on 2.5 minute

cycling at 130% of VO2peak followed by 30-s all-out exercise, glycogen levels were

essentially back to baseline already within 45-75 minutes of passive rest321.

This article is protected by copyright. All rights reserved.


It can therefore be speculated that due to decreased anaerobic glycolysis and

increased oxidative phosphorylation during later bouts of exercise, and perhaps also a faster
Accepted Article
resynthesis of glycogen during the several 10-minute rest periods, the type IIA fibres were

better able to maintain glycogen levels than both type I and type IIX fibres during the

intermittent bouts of ischemic exercise. Importantly, the lactate data reported by Ingemann-

Hansen et al225 from the first 1-2 sets of exercise also provide evidence of recruitment of both

type I and type II fibres types. The ~23 mmol lactate per kg ww in mixed muscle samples

reported after bout 2 is only possible if both fibre types would have been extensively used.

In the only other study to date on glyogen depletion with BFRRE, Cumming et

al21 showed lowered glycogen levels in both type I and type II fibres from the vastus lateralis

one hour after an acute bout of BFRRE. The model training used by Cumming et al21

consisted of 5 sets to failure at 30% of 1RM in the dynamic knee extension exercise, with 45

seconds between sets. The occlusion was achieved with a 135 mm cuff inflated to 90-100 mm

Hg, resulting in about 50-60% reduction of blood flow during rest324. Due to the partial

instead of complete occlusion, and to the short inter-set rest periods, the training model of

Cumming et al21 is therefore more representative of the type of BFRRE that has been

employed in most studies than that of Ingemann-Hansen et al225. Interestingly, although both

fibre types diplayed reduced glycogen at 1 hour post-exercise, the glycogen depletion was

much more marked in the type I fibres, suggesting greater recruitment of these fibres with

BFRRE.

However, the degree of glycogen depletion may reflect not only the intensity

but also the duration or total work, as discussed previously. It is thus possible that type II

fibres were also highly recruited in the study of Cumming et al21, but that the duration of

work was not sufficient to result in a marked glycogen depletion in these fibres, as the type II

fibres would presumably be active mainly during the repetitions with high levels of muscle

This article is protected by copyright. All rights reserved.


activity. After a certain number of repetitions and sets with high activation, the type II fibres

may have dropped out and/or developed less and less force due to fatigue, which would then
Accepted Article
lead to decreased glycogenolytic rates, as shown by Hultman & Sjöholm166. Due to the short

inter-set rest periods (45 seconds) combined with partial occlusion, it is possible that type I

fibres had a relatively faster force recovery between each set in the study of Cumming et al21,

as discussed in Section 4.3. In addition, there may well have been a faster glycogen repletion

rate in the type II fibres after exercise, as noted by Robergs et al214, who demonstrated that

glycogen resynthesis was twice as fast over the first 2 hours post-exercise in type II versus

type I fibres after both low-load (35% of 1RM) and high-load resistance exercise (70% of

1RM) matched for work.

Accordingly, the involvement of type II fibres in BFRRE may have been

underestimated in the study of Cumming et al21. However, an argument against the possibility

of a very fast repletion in type II fibres in this study is that glycogen content continued to be

lower than observed at baseline in both fibre types at 24 hours post-exercise in the BFRRE

leg, although the PAS staining showed a greater decrease in type I fibres over type II fibres at

this time point as well as at 48 hours post. A prolonged depletion is consistent with some

degree of muscle fibre damage and/or remodelling, as has been observed after eccentric

exercise318, and the HSP response reported by Cumming et al21 further supports this notion.

As damage and/or structural remodelling to some degree seemed to have impeded the post-

exercise repletion of glycogen in both fibre types, a greater use of type I fibres during the

acute BFRRE bout appears to be the most plausible explanation for the greater depletion at 1

hour post-exercise in the type I fibres reported by Cumming et al21.

This article is protected by copyright. All rights reserved.


6.2. Studies on phosphocreatine depletion to assess muscle fibre use in BFRRE

Two studies to date have analyzed phosphocreatine levels in biopsies before and after low-
Accepted Article
load BFRRE. In addition to glycogen and lactate, Ingemann-Hansen et al225 also measured

PCr in mixed muscle fibres, and biopsy samples obtained after sets 2-8 showed marked

depletion of PCr, down to about 21% of the baseline level. This 80% reduction exceeds the

61-74% decreases in PCr previously reported in mixed human skeletal (VL) muscle after 12

electrically stimulated high-force contractions at 20 Hz and 50 Hz during concurrent

occlusion167, and approaches the values reported by Söderlund & Hultman169 after 52

ischemic contractions at 20 Hz and by Greenhaff et al52 after 20 ischemic contractions at 50

Hz (both down to ~6-7% of resting values). It has been shown that baseline levels of PCr are

only slightly higher in type II fibres49,52,168. In light of these observations, and given the low-

to-extremely-low post-exercise PCr levels in both fibre types reported by Söderlund et al168

and Greenhaff et al52, there must have been an extensive recruitment of not only type I fibres

but also type II fibres in the ischemic/occlusion exercise study by Ingemann-Hansen et al225,

in line with the glycogen depletion and lactate data discussed above.

Krustrup and coworkers20 investigated the effect of 90 seconds of cyclic knee

extensions with concentric-only contractions, with and without essentially complete

occlusion (300 mm Hg). The knee extension exercise model employed by Krustrup et al20

was the same as that of Andersen & Saltin326, who developed an ergometer for pure

concentric knee extensions with a range of motion between 90 and 160 degrees (180 degrees

= full extension). The load was 29 W and the cadence was 60 extensions per minute,

corresponding to an average joint angular velocity of ~140 degrees/s. With a work period of

90 s, the total number of repetitions thus was around 90. The load in terms of MVC or 1RM

was not reported. Given that the cadence was 3 times faster than in Ingemann-Hansen et al225

with a similar range of motion, and that the work output was 2 times greater, the approximate

This article is protected by copyright. All rights reserved.


load in terms of % of 1RM was probably even lower in Krustrup et al20 than the ~15-20% of

1RM we estimate for Ingemann-Hansen et al225. The fact that the subjects were able to
Accepted Article
complete 90 repetitions at a constant work rate despite 300 mm Hg of occlusion pressure is

also indicative of a work load below 20% of 1RM.

Summarizing their findings, Krustrup et al20 reported that when blood flow was

occluded, fibre recruitment was different from the free flow exercise at the same load as

indicated by lower average PCr concentrations for both type I and type II fibres immediately

after exercise with occlusion, with essentially all fibres having PCr values lower than resting

levels. In contrast, only 2 out of the 5 subjects showed drops in PCr levels in some of the type

II fibres in low-load exercise without occlusion. These results clearly demonstrate that

fatiguing low-load exercise with complete occlusion recruits both type I and type II fibres.

However, the data from Krustrup et al20 also suggest that up to 11% of the type II fibres did

not show evident signs of usage (i.e. no signs of reduced PCr), in contrast to 0% for higher

load exercise (65 W at 60 RPM) performed without occlusion. The percentages of type IIA

and type IIX fibres were reported to be 23 and 20%, respectively.

Thus it appears that during occlusion exercise all three major fibre types were

recruited, including type IIX, although a few type II fibres (10%) showed no signs of use.

Consequently, it may be speculated that the occlusion exercise did not result in sufficient

degree of fatigue to recruit the last remaining type II fibres (possibly of the type IIX type),

but that with a slightly longer duration and continued effort, these would likely had been

recruited as well.

This article is protected by copyright. All rights reserved.


6.3. Summary of glycogen and phosphocreatine depletion in BFRRE

In summary, at least three different studies have provided in essence direct evidence of both
Accepted Article
type I and type II use with low-load BFRRE during varying degrees of occlusion. The results

of two of these studies (Ingemann-Hansen et al225, Krustrup et al20) also strongly suggest that

all fibre types and subtypes can be recruited, from type I to type IIX, although this awaits

definitive confirmation. However, in these studies, the occlusion was complete or at least

severe (300 mm Hg), and as argued previously, only the study of Cumming et al21 used a

partial occlusion training model similar to those typically used in current BFRRE training

studies. As previously discussed however, due to the 1-hour delay between the end of the

BFRRE exercise bout and the biopsy procedure in their study, it is possible that some

glycogen resynthesis occurred especially in type II fibres in the study of Cumming et al21.

It is clear that there is a need for more studies on the patterns and extent of

myofibre recruitment in relation to fibre type composition during BFRRE. PCr depletion

studies may at a first glance seem a better means to assess muscle fibre recruitment than

glycogen depletion studies, as already a few muscle contractions are sufficient to cause some

decrease in PCr99,222,223. However, PCr measurements necessitate that the muscle biopsies are

taken immediately after the exercise has stopped. Glycogen depletion can also provide

important information on muscle fibre use in BFRRE provided that the exercise intensity and

volume are sufficient (e.g. a total of 50-75 repetitions at 20-30% of 1RM) and that biopsies

are taken within minutes after exercise before any significant glycogen repletion has taken

place. Thus, assessments of glycogen depletion have an advantage over PCr depletion with

regard to the allowed delay in muscle biopsy sampling (a few minutes vs a few seconds).

Finally, with the limitations discussed previously in mind, glycogen depletion may also

provide semi-quantitative information on the contributions of the different muscle fibre types

to the total work performed in various BFRRE protocols.

This article is protected by copyright. All rights reserved.


6.4. EMG studies on BFRRE
Accepted Article
6.4.1. Surface EMG

A number of studies have investigated surface EMG responses to various BFRRE protocols

with different exercises, loads, set & repetition configurations and BFR pressures. As surface

EMG is an indirect method of assessesing muscle fibre use253, it is beyond the scope of this

review to list and discuss each of these studies in detail. However, some of the more

influential studies as well as some overlooked pioneering investigations will be discussed in

the following. Studies that contain data which potentially add to the overall picture of muscle

fibre activation patterns and use in BFRRE will also be discussed below.

Person & Golubovich327 appear to have been the first investigators to use

surface EMG to measure muscle activity during low-load ischemic contractions. Their

subjects performed sustained isometric contractions with their elbow flexors to fatigue with a

constant weight of 5 kg (women) or 7 kg (men) with and without vascular occlusion. In the

BFR condition, a pressure cuff was wrapped around the proximal upper arm and inflated to

30-40 mm Hg above the systolic pressure. Without occlusion, the average endurance time

was 9 minutes, which was reduced to 3.5 minutes with cuff occlusion. At task failure, the

integrated EMG amplitude increased to 185% of the initial value without occlusion and to

307% of this value with occlusion. Person & Golubovich327 concluded that artificial ischemia

accelerated and intensified the characteristic increase in EMG amplitude and decrease in

frequency during fatigue. The decline in EMG frequency discussed by Person &

Golubovich327 probably refers to EMG mean power frequency (MPF), which is known to

decrease during fatigue328. The relative load was not specified, but comparisons with an

investigation by Bonde-Peterson et al140 on the impact of occlusion on endurance time at

This article is protected by copyright. All rights reserved.


different loads suggest that the relative load used by Person & Golubovich327 must have been

about 20% of MVC.


Accepted Article
Myers & Sullivan329 also studied the elbow flexors during sustained isometric

contractions to fatigue. Their subjects performed contractions at 25% of MVC, with and

without 300 mm Hg of pressure in a 2.5 cm wide cuff wrapped around the proximal upper

arm. Similarly to the study of Person & Golubovich327, they found markedly reduced

endurance times and increased EMG activity with occlusion, but without specifying the

magnitude of the EMG changes. However, in his PhD thesis, Sullivan330 noted that integrated

EMG amplitude had increased by 542% at the end of the control contraction, and 285% in the

occluded condition, and that overall the EMG results were directly opposite to those of

Person & Golubovich327. The condiderably lower rise in EMG at task failure in the occluded

condition in the study of Sullivan330 is of interest and suggests a decreased excitability of

active myofibres and possibly also neuromuscular transmission failure, in addition to reduced

excitatory drive during their BFR procedure. It is difficult to explain the disparate results of

Person & Golubovich327 and Sullivan330, but they may be related to the specific criteria of

task failure used in the two studies and whether the exercise was performed with support for

the arm or not. The upper arm was supported in the study of Myers & Sullivan329, whereas

Person & Golubovich327 did not report this.

Interestingly, Person & Golubovich327 as well as Myers & Sullivan329 also

studied the effects of occluding the forearm on endurance time and EMG during the isometric

elbow flexor contractions. Both investigations found decreased endurance times, and Person

& Golubovich327 found that EMG at task failure had increased to 256% of the initial value.

Based on the assumption that the forearm muscles did not participate in the task, they argued

that the intensified changes in biceps EMG during conditions of forearm ischemia were

mediated by a reflex and irradiation effects from non-active muscles.

This article is protected by copyright. All rights reserved.


However, it has been estimated that the biceps brachii and brachialis combined

make up only ~57% of the total elbow flexor muscle area and thus that some forearm muscles
Accepted Article
(particularly the brachioradialis) can make substantial contributions to elbow flexor torque331.

Occluding the blood flow to these forearm muscles during a low-load static elbow flexion

contraction would almost certainly reduce endurance time and cause compensatory increases

in muscle activity in the non-occluded biceps and brachialis muscles. Even so, Myers &

Sullivan329 reported decreased endurance times also when the contralateral inactive forearm

was occluded during the task. Sullivan330 noted that EMG increased 411% in this arm. The

cause of this decreased endurance is uncertain, but may be related to group III/IV afferent

input due to metabolite accumulation and/or pressure (see Sections 3.6. and 6.6.).

Signorile et al327 conducted what seems to be the first study on surface EMG in

low-to-moderate load dynamic resistance exercise with BFR. They investigated the effects of

cuff occlusion on EMG in the biceps brachii during elbow flexion exercise performed over a

padded incline bench, at a load of 25RM (established without BFR), which was estimated to

~50-60% of 1RM. Their experimental subjects performed 2 sets of 25 repetitions, one set

with BFR and one set with free circulation, in a randomized order with 10 minutes between

sets. The pace was 3 seconds for the concentric and eccentric phases, respectively, through a

full range of motion with no stops throughout the exercise. A blood pressure cuff inflated to

midway between the diastolic and systolic pressure was used for the occluded condition.

EMG signals appear to have been recorded from the short head of the biceps. The authors

reported an 86% increase in EMG RMS in the concentric phase from the first to the last

repetition with BFR and a 39% increase in EMG from the first to the last repetition in the

non-occluded condition. In the first repetition, the EMG was non-significantly 12% elevated

in the occluded vs. free-flow condition.

This article is protected by copyright. All rights reserved.


The 86% increase in EMG from the first to last repetition reported by Signorile

and coworkers332 is of note. Given that a load of 50-60% of 1RM was used, this suggests that
Accepted Article
the EMG RMS amplitude reached levels similar to those that can be observed with heavy

resistance exercise. The rather large difference in EMG increases between the conditions is

surprising, given that the load was selected to allow only 25 repetitions even without

occlusion. But as the work was matched between the conditions, the effort was almost

certainly submaximal in the free-flow trial as markedly more repetitions can be done in elbow

flexion exercise with free circulation before torque failure is reached8,333, which would

largely explain the observed difference in EMG activity. Alternatively, since the short head

of the biceps is just one of several elbow flexor muscles (cf. discussion above), other elbow

flexor muscles might have fatigued before the short head of the biceps in the non-occluded

condition, limiting the increase in EMG for this particular muscle.

The study of Leonard et al187 has been discussed in Section 4.2., but merits

further mentioning. The subjects performed unilateral plantar flexions against their own

bodyweight on a 40° inclined sliding board, with 40° of knee flexion to emphasise the soleus.

Sets of 21 repetitions in 30 seconds were performed, separated by brief isometric periods

during which soleus H-reflex, M-wave and EMG measurements were obtained. During the

ischemic condition (300 mm Hg, cuff width not stated), the subjects all reached failure during

the sixth set, resulting in a total of ~105-126 coupled concentric-eccentric repetitions in

approximately 3-4 minutes. It should be noted that the ischemic exercise was preceeded by 8-

12 minutes of ischemia, in order to cause a block of proprioceptive afferents but not motor

efferents. Interestingly, during the last two measurement periods before exhaustion, EMG

RMS was drastically decreased (by ~80-85%) while muscle fibre conduction velocity and

MPF were markedly increased. The authors interpreted this as evidence of failure of slow-

twitch motor units and that the increase in EMG MPF was due to compensatory recruitment

This article is protected by copyright. All rights reserved.


of fast-twitch units. This interpretation is supported by the observation of increased muscle

fibre conduction velocity (see Section 5.5.). It is of note that the soleus muscle typically
Accepted Article
consists of ~80-85% type I fibres227,334,335. This type I fibre dominance likely explains why

there were sudden marked increases in muscle fibre conduction velocity with recruitment of

the type II fibres after severe fatigue in the type I fibres in the study of Leonard et al187.

Two influential studies were published 6 years later by Takarada and

colleagues1,18. Both reports had other primary focuses than EMG recording (also assessing

hormonal responses, and muscle strength and mass adaptations), but included EMG results

that were compared between occluded and non-occluded exercise protocols. In the first paper,

Takarada et al18 measured muscle activity in the vastus lateralis during 5 sets of bilateral

dynamic knee extensions at 20% of 1RM to failure combined with partial BFR, with 30-s

inter-set rest periods. This was compared to a work-matched protocol with the same number

of sets and repetitions without BFR. The BFR protocol comprised a 33-mm wide cuff inflated

to 214 mm Hg, which was maintained throughout the entire exercise including rest periods. It

was found that the relative increase in EMG amplitude during exercise with occlusion was

~1.8 times as large as that during exercise without occlusion18. However, the authors did not

report any details about this result, e.g. whether this was measured as overall mean EMG

activity or peak EMG activity.

In the second paper, Takarada et al1 measured muscle activity in the biceps

brachii during sets of unilateral dynamic elbow flexions at 40% and 80% of 1RM with and

without two degrees of partial BFR (50 and 100 mm Hg using a 90 mm wide cuff). The mean

amplitude of the integrated EMG (iEMG) was calculated from the average of the 7th to 9th

repetitions. Takarada et al1 reported that ”without occlusion, the relative iEMG during the

low-intensity exercise was lower by about 40% than that during the high intensity exercise,

indicating that the time-averaged number of muscle fibres recruited was approximately

This article is protected by copyright. All rights reserved.


proportional to the level of force generation. With the increase in occlusion pressure, the

relative iEMG during the low-intensity exercise gradually increased, whereas that during the
Accepted Article
high-intensity exercise was kept unchanged. Consequently, no substantial difference was

observed between the low-intensity and high-intensity exercises at occlusion pressure of 100

mmHg”. However, although 100 mm Hg significantly increased the iEMG at 40% of 1RM, it

appears that the EMG was still ~15% lower than during 80% of 1RM without occlusion.

Nevertheless, Takarada et al1 were the first to show that the EMG levels recorded during low-

intensity BFRRE could approach those observed during conventional high-intensity

resistance exercise.

Also as part of a training study, Moore et al336measured EMG activity during

elbow flexion at 50% of 1RM with and without continuous partial occlusion (100 mm Hg

with a 7 cm wide cuff). They showed a greater relative increase in EMG between the first

repetition in set #3 over set #1 in the BFR arm compared with the non-occluded arm which

performed the same amount of work (~57% vs. ~11%). Further calculations on their data

suggest that EMG activity from the first repetition in the first set to the last repetition in the

last set increased with ~92% and 47% for the BFR arm and the non-occluded arm,

respectively. Collectively, the findings from the studies by Signorile et al332, Takarada et al1

and Moore et al336 thus suggest that agonist EMG levels recorded during dynamic elbow

flexion BFRRE at 40-55% of 1RM can increase with up to ~90% during the the time course

of the exercise session, reaching levels approaching those seen with heavy resistance exercise

using exercise loads ≥80% of 1RM.

Yasuda et al180 compared the effects of four different degrees of BFR on

relative iEMG during a standardized elbow flexion exercise protocol at 20% of 1RM

consisting of 30-15-15-15 repetitions in sets 1-4. Occlusion pressures were 0 (free-flow

condition), 98, 121 and 147 mm Hg, respectively. The cuff was 30 mm wide and the dynamic

This article is protected by copyright. All rights reserved.


elbow flexion exercise was performed over a 45° incline bench, with each complete repetition

taking 2.4 s, with 30 s rest periods between the sets. In addition to iEMG during the exercise
Accepted Article
bouts, MVC and iEMG during MVC were measured before and 1 and 2 minutes post-

exercise, with maintained BFR during the 1-minute post MVC and free circulation during the

2 minute post MVC. From the first repetitions in the first set to the last 5 repetitions of the

fourth set of the BFRRE bouts, iEMG increased by ~90-135%, with the greatest increases

seen with 147 mm Hg and the lowest with 98 mm Hg. In the free-flow condition, iEMG

increased by only ~60%. MVC measured at 1-minute post decreased in the same order, i.e.

the largest decrements resulted from BFRRE at 147 mm Hg (~38% reduction) and the least

from free-flow exercise (15% reduction). Interestingly, the same order was also observed in

the iEMG amplitude obtained during 1-minute post-exercise MVCs: ~44% reduction at 147

mm Hg, ~32-34% at 121 and 98 mm Hg, and 10% with free circulation.

In a follow-up study, Yasuda et al181 compared the effects of varying degrees of

BFR on iEMG during 3 different elbow flexion exercise protocols at 20% of 1RM: 1 x 30

repetitions (Experiment 1), 3 x 10 repetitions (Experiment 2) and 30-15-15-15 repetitions

(Experiment 3). In Experiment 1, in two conditions restriction pressures of 160 mm Hg

(”moderate restriction”) and 300 mm Hg (”complete occlusion”) were applied, respectively,

while the third condition used free-flow circulation (control). In Experiment 2, the same three

conditions were used. In Experiment 3, the two conditions were 160 mm Hg and free

circulation. Cuff dimensions and the elbow flexion exercise were the same as in the previous

study. In addition to iEMG amplitude during exercise, MVC and iEMG activity during MVC

was measured before and 1 and 2 minutes post-exercise, as in the previous study. In

Experiment 1 and 2, exercise with complete occlusion tended to induce in the greatest

increases in iEMG from the first to the last repetitions (up to ~130%). In Experiment 3, the

highest elevation in iEMG was seen in exercise with moderate restriction (~125%). With the

This article is protected by copyright. All rights reserved.


most fatiguing protocols with moderate restriction and complete occlusion, the decrements in

MVC and iEMG were even greater than in the previous study, with iEMG during 1-minute
Accepted Article
post MVCs decreasing to as low as 40-50% of pre-exercise iEMG.

The EMG activity recorded during BFRRE by Yasuda et al180,181 showed

average values during the final repetitions in the most fatiguing protocols that were about 2.3

times higher than in the first repetitions. This magnitude of change is surprisingly low, as one

would expect a greater increase given that the load was only 20% of 1RM and that the ratings

of perceived exertion after the final repetitions in set 4 were 17-18 on a Borg 6-20 RPE scale,

where 17 is ”very hard”, 19 corresponds to ”extremely hard” and 20 is ”maximal exertion”

(Borg332). In a later study using almost identical methods, Yasuda et al333 reported much

greater increments in EMG activity throughout the period of acute BFR exercise. In this latter

investigation, Yasuda et al333 compared elbow flexion exercise protocols to failure with and

without BFR at 20% of 1RM. In the BFR condition, the EMG values at concentric failure

were up to 3.9 times higher than during the first repetitions. In the non-occluded trial, EMG

increased by up to 4.4-fold. Even these increases suggest a submaximal EMG level in the

highest repetitions during BFRRE compared to free-flow MVC conditions, given the low

loads used and a curvilinear EMG rise with increasing force, (see Section 5.3.1.). For

comparison, the data in Christie et al67 and Moritani & Muro256 suggest that in free flow

conditions EMG RMS activity in the biceps brachii increases ~10-fold from 20% of MVC to

100% of MVC.

The 1-minute post-exercise MVCs after BFRRE with ”moderate restriction” in

Yasuda et al180,181 reveal 45-50% reductions in EMG compared with pre-exercise MVCs, and

almost 60% decrements after BFRRE with complete occlusion. This observation may provide

insight into why EMG did not increase more during dynamic BFRRE at 20% of 1RM. The

45-60% reductions in post-exercise EMG are of a magnitude which is unlikely to be

This article is protected by copyright. All rights reserved.


explained by central fatigue alone, which appears to be limited during occluded exercise of

relatively short durations as judged by interpolated twitch measurements338,339. Thus, more


Accepted Article
likely contributing factors would be decreased motor unit firing rates due to spike-frequency

adaptation and inhibitory group III/IV afferent input. Lower firing rates in some motor units

during fatiguing exercise, as observed by Garland et al139, could result in lower EMG even if

no fibres were derecruited or dropped out due to propagation failure. However, we suggest

that the submaximal exercise and post-BFRRE EMG amplitudes reported in the studies of

Yasuda et al180,181 may reflect a markedly reduced excitability of the muscle fibres, and

possibly some degree of NMJ transmission failure (see Section 4.2., and 6.4.2. below).

Wernbom et al19 investigated EMG responses in the vastus lateralis and

medialis muscles during three sets of unilateral low-load knee extensions to failure at 30% of

1RM with and without BFR (100 mm Hg with a 13.5 cm cuff). In line with earlier results

with near-complete occlusion at 200 mm Hg (Wernbom et al314), the partial occlusion

reduced the number of repetitions compared to free-flow conditions by ~31% in the first set.

High EMG levels were demonstrated in both conditions, with concentric EMG increasing up

to 2.7-2.8 times from the first to the last repetitions (up to 94-102% of MVC EMG), but with

no significant differences regarding maximal concentric muscle activity between the

conditions. Interestingly, EMG increased markedly during the eccentric phase in both

conditions, up to 2.2-fold and 2.5-fold respectively, with a higher EMG in set 3 for the free-

flow condition. Wernbom et al19 were the first to report EMG increases during not only the

concentric but also the eccentric contraction phases during acute bouts of BFRRE. In

addition, it was the first study to compare dynamic low-load training to failure with and

without BFR.

In our experience, very low-load unilateral BFRRE (~20-25% of 1RM)

performed to fatigue results in a more or less hyperbolic increase in EMG RMS amplitude as

This article is protected by copyright. All rights reserved.


the point of concentric failure draws close (an example is shown in Figure 5a, from

Wernbom340). Similar observations have been reported by others (Farup et al8, their Fig.6).
Accepted Article
This appearance is remarkably similar to the EMG curves reported in the studies on low-force

isometric contractions (10-40% of MVC) sustained to fatigue by Vestergaard-Poulsen et

al308,309 and Houtman et al311, discussed in Section 5.5. The noticeably steeper increase in

EMG RMS amplitude in the vastus lateralis and medialis during the last 6 repetitions in the

first set may indicate an additional recruitment of large superficial motor units (presumably

innervating type II myofibres). Note that EMG activity did not seem to reach quite as high

during low-load BFR knee extensions (Figure 5a-d) as during heavy load knee extensions

without BFR (Figure 6)

Investigations by others have confirmed that EMG increases during the eccentric phase in

fatiguing BFRRE and that quadriceps EMG can reach high levels during fatiguing very low-

load knee extensions (20-30% of 1RM) irrespective of BFR341. Notably, however, available

data suggest that EMG amplitudes in the quadriceps at these loads may not reach the levels

seen with high-load (70-75% of 1RM) resistance training341,342, even when low-load training

is performed to failure without occlusion341. In the study of Loenneke et al341, EMG during

the concentric phase of BFRRE at 20% of 1RM did not exceed 60% of MVC in any of the

different BFR conditions, and EMG recorded during 20% of 1RM to failure without BFR

also did not exceed 60% of the MVC EMG activity. The increases in the concentric phase

were 114% in the 20% of 1RM to failure protocol and 45-82% in the BFRRE at 20% of

1RM.

Somewhat in contrast, Fatela et al342 reported that EMG RMS amplitude

increased up to ~100% of MVC in the rectus femoris and ~85% of MVC in the vastus

medialis by the end of 4 sets of knee extensions at 20% of 1RM. However, EMG activity at

This article is protected by copyright. All rights reserved.


the start of the exercise was unusually high (~50% of MVC), which is puzzling considering

the very low exercise load (20% 1RM). The exercise was performed with concentric-only
Accepted Article
repetitions in an isokinetic dynamometer set in the isotonic mode, with 1 s concentric phases

and 1 s rest between repetitions. Another difference was that knee extensions were performed

unilaterally instead of bilaterally as in Loenneke et al341. Interestingly, high-load knee

extensions at 75% of 1RM without BFR were also investigated by Fatela et al342, in which

quadriceps EMG values reached ~125% of MVC in the repetitions with the highest muscle

activity. This is somewhat similar to the findings of Amiridis et al270 and Beutler et al273,

discussed in Section 5.3.1. This increase could be due to increased motor unit recruitment

and/or elevated firing rates and/or changes in muscle fibre excitability during maximal

dynamic compared with isometric contraction conditions.

In summary, evidence suggests that EMG activity during BFRRE with loads of

40-55% of 1RM can reach levels close or comparable to those encountered with high-load

resistance exercise, at least when compared in the final repetitions of the respective exercise

sets. In contrast, yet other study reports indicate that during BFRRE with very low loads

(~20% of 1RM), peak EMG RMS levels may fall short of those seen with conventional heavy

strength training, particularly in the case of BFR training involving the elbow flexors. It is

hypothesised that this discrepancy is mainly because of the effects of decreases in firing rates

and/or muscle fibre excitability, and in addition potentially transmission failure in some

muscle fibres.

6.4.2. Intramuscular EMG

Magora et al343 studied the effects of cuff ischemia on motor unit recruitment and activity in

the biceps brachii in a low-load isometric endurance task. The subjects performed a sustained

isometric contraction against a counterload of ~20% of MVC until complete inability to

This article is protected by copyright. All rights reserved.


sustain target force. The task was performed first with free flow and then with cuff occlusion,

with 1 hour of rest in between. Before the ischemic contraction, a pressure cuff (width not
Accepted Article
reported) was inflated to 30 mm Hg above the systolic blood pressure. Occlusion reduced the

time to task failure by 72.5% on average (81 vs. 296 seconds). Interestingly, in the occluded

condition, there were greater numbers of large spike amplitudes and fewer low-amplitude

spikes in the EMG signal recorded at the beginning of the task. The authors concluded that

the contraction was initiated involving a higher proportion of large motor units. Curiously

though, after the initially high levels, the mean maximal spike amplitude decreased and low

amplitude spikes increased in the ischemic contraction, which was opposite to that observed

in the non-ischemic contraction. At task failure, the mean maximal spike amplitude was very

similar between the conditions.

Magora et al343 discussed the decrease in mean maximal spike amplitude during

the ischemic contractions as ”the gradual transformation of big motor units to small”. It is

unclear whether this refers to a partial loss of active muscle fibres in large motor units, thus

effectively rendering active motor units smaller, or the result of a more complete drop-out of

bigger motor units. Regardless, a drop-out of some muscle fibres due to neuromuscular

transmission failure (Section 4.2.) could explain the observed decreases in spike amplitudes.

Moritani et al17 investigated exercise with BFR using both surface and

intramuscular EMG. The subjects performed repeated hand grip contractions at 20% of MVC

for 2 s followed by 2-s rest for 4 min with either free blood circulation or arterial occlusion

given between the 1st and 2nd min (200 mm Hg on the upper arm, cuff width not reported).

The intramuscular motor unit spikes and surface EMG data indicated that mean motor unit

spike amplitude, firing rates, EMG RMS and MPF remained constant throughout the

experiment with unhindered circulation. In contrast, significant increases in mean motor unit

spike amplitude and frequency were evident during the contractions with arterial occlusion,

This article is protected by copyright. All rights reserved.


along with progressive increases in surface EMG RMS amplitude and decreases in MPF. The

authors interpreted the elevations in spike amplitude and frequency as suggesting new motor
Accepted Article
unit recruitment and an increased discharge rate of relatively high-threshold units.

Notably, mean spike amplitude in Moritani et al17 had increased by ~40%

during the last 15 seconds of the occlusion and at this point EMG RMS was elevated by

about 90%. The gradual increase in both parameters would appear to reflect fatigue-induced

compensatory recruitment, although altered motor unit activation patterns resulting from

pressure-induced stimulation of cutaneous receptors and other mechanically sensitive

afferents cannot be ruled out (see Section 6.6.).

6.5. 31P-MRS studies on muscle fibre recruitment in BFRRE

As with other types of exercise, the occurrence of ”split” Pi peaks in 31P-MRS spectra has

been used to infer type II fibre recruitment with strenuous BFRRE9,10,344-346. In these studies,

a unilateral plantar flexion training model was employed, involving the triceps surae muscles

(the gastrocnemius medialis and lateralis, and the soleus) and consisting of coupled

concentric and eccentric contractions. In one of the first studies on BFR resistance-type

exercise and muscle fibre recruitment, Yoshida & Watari344 reported split peaks during

occluded but not during free-flow exercise to exhaustion, suggesting recruitment of type II

fibres in the former mode of exercise but not the latter.

However, since they reported neither the workload in watts nor the absolute

load in kilograms (or relative load in % of 1RM), and due to other uncertainties in the

description of the exercise (e.g. range of motion was not noted), it is difficult to assess

relative exercise intensity in this study. More importantly, the 80 mm surface coil was placed

over the center of the calf muscle (i.e. between the gastrocnemius medialis and lateralis

muscles), which would almost certainly mean that both the individual muscle components of

This article is protected by copyright. All rights reserved.


the gastrocnemius were sampled, increasing the risk that the detection of split Pi peaks was

caused by contamination artifacts due to the non-localized 31P-MRS spectra rather than due to
Accepted Article
recruitment of fresh type II muscle fibres. In principle, the fact that at least two different

muscles were sampled limits the conclusions pertaining to the pattern of motor unit

recruitment that can be derived from the study of Yoshida & Watari344.

In a series of studies, Suga, Okita, Takada and colleagues9,10,345,346 investigated

low-load plantar flexion exercise combined with BFR. They used mostly an intensity of 20%

of RM, although loads of 30% and 40% of 1RM with BFR were also studied, as well as 65%

of 1RM without occlusion. BFR was achieved using a cuff around the thigh, and the

combination of very wide 18 cm cuffs and ~143-150 mm Hg of pressure was likely sufficient

to cause near-complete occlusion in their supine test position (see Crenshaw et al313), as also

indicated by the very limited recovery in PCr observed during the rest periods between sets

(see Suga et al9). Similarly to Yoshida & Watari344, split Pi was observed albeit to varying

degrees in the different studies. However, it is not immediately obvious whether the split Pi

peaks reported in the studies of Suga and colleagues9,10,345,346 truly reflected the initial

recruitment of type II fibres (type IIA) in addition to already active type I fibres, as the split

Pi peaks could also mark the recruitment of type IIX and/or IIAX fibres in addition to already

active type IIA fibres, along similar lines as the suggestions put forth by Vandenborne et

al284, (cf. Section 5.4.). This possibility was not discussed explicitly in the studies of Suga et

al9,10,345,346.

Notably, even the high load free-flow protocol (65% of 1RM) which was

investigated along with the BFRRE protocols in these studies9,345,346 did not yield dual Pi

peaks in all subjects, despite each exercise set consisting of 30-60 repetitions, for a total of

60-90 repetitions per session. This is remarkable, as is the fact that the subjects performed up

to 60 repetitions per set, since a load of 65% of 1RM would be expected to recruit type IIA

This article is protected by copyright. All rights reserved.


fibres already from the start of the exercise while also involving type IIAX and IIX fibres to

become active not long after. In comparison, Trappe et al347 reported that their subjects were
Accepted Article
able to perform only about 15 repetitions of unilateral plantar flexion at a load of ~70% of

1RM. Notably, the subjects in the paper of Trappe et al347 performed standing unilateral

plantar flexions with bodyweight (80 kg) and up to 10 kg of extra weight, to compare with 33

kg in supine unilateral plantar flexion for the male subjects employed by Suga et al9.

These puzzling discrepancies in load-repetition characteristics between studies

may in part be explained by differences in posture (supine vs upright) and range of motion

(ROM). Available data suggests that plantar flexion torque decreases in an essentially linear

manner with increasing plantar flexion angles, so that low-velocity concentric torque at 30

degrees of flexion (0 degrees = neutral position) is only about 25-30% of that at -10 degrees

of flexion348-350. Accordingly, Gravel et al348 concluded that the maximal strength capacity of

the plantar flexors is best represented by the ankle extensor moment measured in slight

dorsiflexed joint angles when the muscles are at elongated length. In the studies of Suga and

colleagues, the 1RM was determined concentrically as the highest weight the subject could

lift completely throughout a limited ROM (~5 cm). This probably means that the 1RM was

mainly dependent on the torque capacity at the end of the ROM, which would be

considerably lower than at the start of the movement. It is also of relevance that concentric

plantar flexion torque is markedly enhanced by preloading350 and by a preceding stretch-

shortening cycle even at submaximal loads351.

Therefore, it is likely that the plantar flexion movement employed in the studies

of Suga and colleagues9,10,345,346 was underloaded during a large part of the ROM, thus

requiring much less effort and recruitment during this part than at the end range of the

movement. In this scenario, type II fibres would not necessarily be recruited from the start of

the exercise even at a nominal load of 65% of 1RM, except for perhaps a brief activation

This article is protected by copyright. All rights reserved.


during the last degrees of concentric plantarflexion. This would in part explain that the

subjects were able to perform as many as 60 repetitions per set with 65% of 1RM, apparently
Accepted Article
without reaching exhaustion, and how the same subjects could perform a total of 90-120

repetitions during near-complete occlusion at 20% of 1RM, also seemingly without reaching

concentric contraction failure.

The end-exercise pH values after sets of BFRRE with near-complete occlusion

at 20% of 1RM in these studies9,10,340,341 were between ~6.84 and 6.92, with a resting pH of

about 7. Interestingly, in the studies where multiple sets of repetitions were employed at 20%

of 1RM9,10, end-exercise pH values clearly tended to be lower than in the studies using only a

single set protocol340,341: ~6.84 vs 6.91-6.92. Moreover, the percentages of subjects

displaying split Pi peaks were much greater in the studies with the lower end-exercise pH

values: 64-75% vs 31-33%. Notably, end-exercise pH in the range of 6.6-6.7 have been

reported for the gastrocnemius muscle after strenuous plantar flexion exercise295,300 and 6.7

after BFRRE at 30% of 1RM352. Therefore, pH values may well be expected to drop to ~6.6–

6.7 and possibly even lower in the gastrocnemius muscles following low-load BFRRE

protocols performed to torque failure. Given the greater incidence of split Pi peaks in these

studies once the mean pH levels dropped below 6.9 (discussed above), recruitment of type II

fibres may have occurred once pH dropped within the range of ~6.8-6.9 in the BFR plantar

flexion exercise model of Suga and colleagues9,10,345,346, suggesting that this response

reflected the firstly recruited population of type II fibres (i.e. type IIA).

Nevertheless, some puzzling observations are not easily explained by the above

scenario. For example, some of the subjects in the study of Suga et al346 were able to perform

as many as 60 successive repetitions in the high-intensity resistance exercise protocol during

free-flow conditions without displaying split Pi, and they also managed to perform 60

repetitions during near-complete occlusion at 20% of 1RM, also without generating visible

This article is protected by copyright. All rights reserved.


split Pi peaks. However, most of these subjects displayed split Pi peaks after 60 repetitions at

40% of 1RM with BFR, which was the most PCr depleting protocol. Therefore, it seems
Accepted Article
possible that the surprisingly late split Pi peaks observed by Suga et al346 in these subjects

were actually signifying recruitment of type IIX and/or IIAX fibres, rather than IIA fibres. In

this scenario, type IIA fibre recruitment may not have been detected for at least two possible

reasons: a relatively small pure type IIA pool in these individuals and/or only minor

differences between type I (including type I-2) and type IIA fibres in terms of intracellular

lactate accumulation.

In this context, it is noteworthy that although biopsy studies with myosin

ATPase stainings have shown that the gastrocnemius medialis and lateralis muscles are

composed of ~40-50% type II fibres227,259,334,335,353, results from a study which used a

staining method for lipid droplets and mitochondria indicate that the gastrocnemius muscles

consist of as much as 82-84% mitochondria-rich fibres, 8-9% intermediate fibres and 7-10%

mitochondria-poor fibres38. While the latter two fibre phenotypes might correspond to type

IIAX and type IIX, the discrepancy between the 50-60% type I fibres reported for the

gastrocnemius in the aforementioned studies and the large proportion (82-84%) of

”mitochondria-rich fibres” reported by Buchthal & Schmalbruch38 strongly suggests that a

considerable portion of type II fibres, presumably mostly type IIA fibres, are rich in

mitochondria and lipid droplets to an extent that approaches type I fibres. This notion is in

line with the overall slow contractile properties of the gastrocnemius reported by these same

authors38 and also with the observation that FR units (presumably consisting of type IIA

fibres) in the human gastrocnemius medialis have a fatigue resistance profile resembling that

of S units, which consist of type I fibres38.

With regard to the distribution of type II fibre subtypes in the gastrocnemius, a

biopsy study on the gastrocnemius lateralis (GL) muscle in habitually physically active

This article is protected by copyright. All rights reserved.


subjects showed only 3% type IIX fibres, while type IIA and type I fibres comprised 38% and

59% of the myofiber pool, respectively, as judged by ATPase staining analysis354. A study on
Accepted Article
fibre type distribution in the GL in young untrained but healthy subjects report that the GL

contained 59.4% type I fibres and 26.4% type IIA fibres, with the remaining ~14% classified

as type IIX fibres also using ATPase staining355. Assuming that both type I and type IIA

fibres in the human gastrocnemius are rich in mitochondria and lipid droplets, and that some

of the type IIX fibres in Vandenborne et al355 were in reality type IIAX, the data reported by

Vandenborne et al355 seem to match those of Buchthal & Schmalbruch38 reasonably well.

Collectively, this information suggests that type IIA is the most common type II

subtype in the human gastronemius in young healthy subjects and that type IIA fibres in the

gastrocnemius muscle are nearly as fatigue-resistant as type I fibres in the same muscle,

probably at least in part due to their abundance of mitochondria and lipid droplets. In turn,

this similarity may be of considerable significance for the balance between aerobic and

anaerobic metabolism during exercise. Specifically, it may be that the differences in lactate

accumulation and associated drops in pH between type I and type IIA fibres in the

gastrocnemius during exercise are relatively minor, thus making it difficult to detect split Pi

peaks in the 31P-MRS spectra caused by the recruitment of type IIA fibres in addition to

already active type I fibres.

We therefore speculate that the Pi splits reported in the studies of Suga and

colleagues9,10,345,350 may to a large extent reflect a progressive recruitment of type IIAX

and/or type IIX fibres, rather than type IIA fibres. This possibility is further indictated by the

observation in the study of Suga et al346 that the RPE at the end of the BFRRE protocol (1 set

of 60 repetitions at 20% of 1RM) was 6.6 out of 10 on the Borg CR-10 scale (i.e. close to

”very strong”), despite that split Pi was observed in only 31% of the subjects. In comparison,

the high load protocol (1 set of 60 repetitions at 65% of 1RM) resulted in split Pi peaks in

This article is protected by copyright. All rights reserved.


70% of the subjects and an RPE of 8.2. The most strenuous BFRRE protocols at 20% of 1RM

in the studies of Suga and Takada et al9,10 resulted in Pi splits in 64-75% of the subjects.
Accepted Article
However, an important limitation of the studies of Suga et al9,345,346 and Takada

et al10 is that the exact position of the MRS surface coil used to detect the metabolic changes

was not reported, the only information provided in all of the reports was that ”an 80-mm

surface coil was placed under the muscle belly of the right gastrocnemius”. If the 80 mm

surface coil was placed between the gastrocnemius medialis and lateralis muscles, as in

Yoshida & Watari344, it would probably mean that all these studies are prone to the same

criticism as the report of Yoshida & Watari344 with regard to the potential effects of signal

contamination due to non-localized 31P-MRS spectra.

Regardless, the marked gains in both muscle strength and CSA of the plantar

flexors following 4 wks of BFRRE reported by Takada et al10 are noteworthy given the very

low exercise loads used in this study, in actuality probably closer to 10-15% of 1RM rather

than the reported 20% of 1RM. This raises interesting questions regarding the minimum load

in BFRRE that would be sufficient for inducing muscle hypertrophy and functional strength

gains.

6.6. Can muscle fibre recruitment order be changed during cuff occlusion?

Some authors have suggested that the normal order of activation of muscle fibres (type I 

type IIA  type IIX fibres according to the Henneman size principle of motor unit

recruitment) may be altered during exercise with occlusion, so that type II fibres could be

activated preferentially over type I fibres356,357. To the best of our knowledge, at least four

studies have reported indications of possible alterations in the recruitment order during

occluded blood flow, although being observed during relatively long-lasting ischemia (~20-

30 minutes) or with ischemia of unspecified durations108,191,343,358.

This article is protected by copyright. All rights reserved.


In their study on impulse transmission in the extensor digitorum communis

(EDC) muscle in humans with needle electromyography, Dahlbäck et al191 observed that
Accepted Article
”during ischemia the recruitment pattern of the motor units altered so that those formerly only

recruited at stronger contraction were now the first to be recruited”. Dahlbäck et al191 used a

blood pressure cuff (width not reported) on the upper arm inflated to 200 mm Hg to induce

ischemia, and instructed their subjects to maintain contractions with steady motor unit firing

rates of between 2-15 Hz. Probably because their main focus was neuromuscular

transmission, they did not further specify the strength of the contractions used and the

approximate level of effort that was required to recruit the higher-threshold units prior to

ischemia, nor did they report the time of ischemia when the reported alterations took place.

Nevertheless, it was stated that the experiments were performed on motor units which could

be activated even at the slightest contraction of the muscle. Furthermore, data in Monster &

Chan58 obtained in the EDC muscle show that the first recruited units can reach ~16 Hz

already at a few percent of the MVC. Taken together, this suggests that the contractions used

by Dahlbäck et al191 involved very low forces even when motor unit firing rates were 10-15

Hz.

Grimby and Hannerz studied motor unit recruitment order and firing rate ranges

during ischemia in the tibialis anterior and the extensor digitorum brevis muscles using fine

wire and needle electrode recording (Grimby & Hannerz358; Hannerz & Grimby108). A

pressure cuff (width not stated) was applied around the thigh and inflated to higher than the

systolic blood pressure, to ~140 mm Hg. The sensory and motor functions of the lower leg

successively disappeared over a time course of 20-40 minutes. It appears from the description

in Hannerz & Grimby108 that the occlusion was not quite complete, as they chose a pressure

which induced a partial nerve blockade in order to lengthen the period during which the

motor responses could be studied. In the first study, Grimby and Hannerz358 investigated

This article is protected by copyright. All rights reserved.


recruitment changes in the tibialis anterior during partial ischemic nerve blockade with

dorsiflexor test contractions at 10-20% of MVC for 20-30 minutes. At 15-20 minutes into the
Accepted Article
ischemic period, an altered order of motor unit recruitment was observed, in that increasingly

more other units became recruited before the unit which had the lowest threshold in free-flow

conditions. By 25 minutes, a few high-frequency units also had lower thresholds than this

unit. Presumably, these were high-threshold units, as they were not activated during test

contractions at 10-20% of MVC performed after the ischemic blockade had ended.

In their second study, Hannerz and Grimby108 focused their attention mostly on

motor unit firing rate changes during ischemia, but made some additional observations on

recruitment thresholds. While most intermittently firing units did not change their firing

properties during ischemia, three high-threshold units in the tibialis anterior which normally

were recruited above 75% of MVC became recruited at substantially lower absolute and

relative tensions upon ischemic blockade. In one dramatic example, a very high threshold

unit which was normally recruited at 88% of MVC became active already at 4% of the pre-

fatigue MVC during ischemic conditions, when the maximum tension had dropped to 32% of

the baseline MVC. The drastic decrease in recruitment threshold strongly suggests that for

this particular unit, an almost complete reversal in the normal recruitment order must have

taken place.

The findings of Magora et al343 (discussed previously in Section 6.4.2.) of

greater numbers of large EMG spike amplitudes and fewer low-amplitude spikes in the

beginning of an ischemic low-load endurance task may also be interpreted as evidence of

deviations in recruitment order. At a first glance, the increase in the average amplitude of the

motor units during the early part of the ischemic contractions seems quite large (72%).

However, the range of motor unit amplitudes that can be observed in the biceps brachii

during an MVC suggests on the order of ~30-fold differences between the smallest and the

This article is protected by copyright. All rights reserved.


largest amplitudes (see e.g. Moritani et al279). Data on motor unit amplitude in relation to

recruitment threshold in the biceps brachii during free-flow conditions in the paper of
Accepted Article
Gydikov & Kosarov109 suggests that a 72% increment in spike amplitude over those of motor

units recruited at 20% of MVC would correspond to units recruited at ~30% of MVC.

Furthermore, using multiple indwelling electrodes, Buchtal et al359 reported that during free-

flow conditions the amplitudes of successively recruited motor units in the biceps brachii

increased so that the secondly recruited units in their recordings on average demonstrated a

98% greater EMG amplitude than the firstly recruited units. The firstly recruited motor units

were active at very weak contraction efforts, less than required to support the weight of the

forearm.

Taken together, these observations indicate that if recruitment order changes

due to cuff occlusion occurred in the study of Magora et al343, they were still modest, possibly

on the same order as those observed in Masakado et al76 or less. It should also be noted that

the recruitment order of different motor units was not directly investigated in the study of

Magora et al343, and reversals in the recruitment order were therefore not definitely proved or

disproved in their study.

As discussed above, alterations in the motor unit recruitment order during cuff

ischemia might be caused by blockade of afferent inflow to the CNS358, intramuscular pain74,

and enhanced cutaneous inputs61,75; and ischemia can also impact on the activation of

individual myofibres in a given motor unit via effects on the motor end-plates and muscle

fibre excitability and/or motor nerve axons (Section 4.2.). In addition, the magnitude of

applied pressure can affect alpha-motorneuron excitability per se. Specifically, application of

limb pressure led to decreased Hoffman-reflex (H-reflex) responses both in a massage

setting360 and when administered as circumferential pressure via air splints or pressure

cuffs361,362. The applied pressures in these investigations were 19-28 mm Hg in the study of

This article is protected by copyright. All rights reserved.


Goldberg et al360 and 37-45 mm Hg in Robichaud et al361 and Agostinucci362. In contrast,

Agostinucci363 found no decrease in the soleus F-wave response with 45-50 mm of pressure.
Accepted Article
Agostinucci363 suggested that this difference reflected the different types of motor units

involved in the two responses, with the H-reflex primarily recruiting smalller motorneurons,

while the high-stimulus F-wave (supramaximal stimulation) may preferentially recruit larger

faster conducting motorneurons. If this scenario is true, it suggests the interesting possibilty

that alterations in recruitment order due to applied limb pressure may occur also in voluntary

muscle contractions.

If the occlusion is applied just before onset of contraction, as appears to have

been the case in the study of Magora et al343, partial reversals due to loss of some small motor

units seem unlikely, as the duration of fatigue would likely not have sufficient time to affect

the axonal branches, the motor end-plates and/or the excitabiliy of the slow fibres. In this

scenario, a facilitation of larger motor units arising from the cutaneous stimulation of the cuff

over the working muscle would seem a more logical explanation, in line with the study of

Garnett & Stephens61. An influence from group III/IV afferents would at a first glance seem

improbable to cause such rapid effects, as the firing of these afferents is known to be

stimulated by metabolite accumulation, which in turn requires a certain time to develop.

However, group III/IV afferents have also been shown to be very sensitive to pressure,

significantly more so than to stretch, and there is a linear relationship between the level of

pressure applied and the response of these afferents364. The alterations in motor unit activity

in the study of Magora et al343 might thus be explained by the placement of the cuff over the

biceps muscle belly and its effects on the inflow from cutaneous and/or group III & IV

afferents onto spinal alpha-motorneurons, with a consequent inhibition of smaller motor units

and a facilitation of larger units.

This article is protected by copyright. All rights reserved.


In contrast, the PCr and glycogen depletion data from the studies of Ingemann-

Hansen et al225, Krustrup et al20 and Cumming et al21 (discussed in detail above) are if
Accepted Article
anything consistent with an orderly recruitment of motor units. If any major departures from

the size principle occurred in these studies, this should have been reflected in greater

depletion of PCr and glycogen in type II fibres compared with type I fibres. Instead, an

overall tendency for the expected result can be seen, i.e. a greater use of type I fibres.

Altogether, the available evidence suggests that the size principle for motor unit recruitment

is largely intact during dynamic BFRRE and that if reversals occur, they are relatively minor

(only affecting a few units) and/or of temporary nature. The muscle activation patterns

observed by 31P-MRS analysis and EMG recordings are consistent with this scenario. If

systematic reversals or at least major deviations take place during dynamic BFRRE, these

would be expected to show up in the 31P-MRS spectra in the form of a lower pH Pi peak of

type II fibres appearing at the same time or even before the higher pH Pi peak of type I fibres,

due to the fast activation and rate of glycolysis in type II fibres. This has not been the case

(discussed in detail above). Moreover, the observation by Leonard et al187 of markedly

greater muscle fibre conduction velocites (signifying type II fibre recruitment) near the point

of exhaustion during low-force ischemic exercise in the soleus muscle is also consistent with

a largely intact orderly recruitment. Importantly, this was the case in spite of a probably

blocked proprioceptive inflow.

7. Conclusions and implications.

7.1. Order of muscle fibre recruitment in BFRRE

Of the various methods of investigating muscle fibre activation which have been reviewed

here, only glycogen and PCr depletion studies can provide essentially direct evidence of fibre

This article is protected by copyright. All rights reserved.


recruitment and usage during acute BFRRE, although these methods are not without caveats.

However, complementary information may be derived from EMG and 31P-MRS studies
Accepted Article
involving various BFRRE protocols. Studies with intramuscular EMG electrodes have

reported indications of alterations in the recruitment order during low-load contractions

combined with cuff occlusion, but these have generally been observed with isometric

contractions performed after many minutes of ischemia, with the possible exception of the

study of Magora et al343. Even under prolonged ischemic conditions, the extent of motor unit

recruitment alterations seems minor, and the relevance of these observations to BFRRE is

uncertain. The data from PCr and glycogen depletion studies involving BFRRE are consistent

with a largely intact order of motor unit recruitment, and indirect evidence from 31P-MRS and

EMG investigations further support this notion. In other words, there are no indications of

any major departures from the Henneman size principle for motor unit recruitment in low-

load dynamic resistance exercise using concurrent cuff occlusion.

7.2. Patterns of muscle fibre use and fatigue in BFRRE

The evidence reviewed strongly suggest that although type II fibres (including type IIX) can

be recruited with fatiguing low-load BFRRE, type I fibres are used to a greater extent, as

judged by both glycogen and PCr depletion data. In a previous paper, we suggested a

scenario where the type I fibres become highly fatigued during a first set of unilateral BFRRE

at 30% of 1RM, thus necessitating the recruitment of an increasing number of motor units

containing type II fibres as the exercise progresses. We here extend this model by proposing

that all subtypes of type II fibres (type IIA, type IIAX and type IIX) can be recruited during

low-load BFRRE even at 20-30% of 1RM if the work duration and degree of fatigue is close

to maximal. The suggested scheme for the recruitment of motor units during fatiguing

BFRRE is depicted in Figure 7.

This article is protected by copyright. All rights reserved.


In addition, we previously proposed that type II fibres will have to be recruited

earlier in a second and a third set than in the first set, presumably because of residual fatigue
Accepted Article
in type I fibres19. Importantly though, the typically short inter-set rest periods in BFRRE and

the differences in metabolite and force recovery between the fibre types (including subtypes)

will undoubtedly influence the specific fatigue patterns. Available evidence shows that if all

sets are performed to failure, the number of repetitions will drop for each set, but especially

between the first and second sets. For example, Wernbom et al178,324 reported that with 30%

of 1RM and 45 s rest periods, the number of repetitions performed before concentric torque

failure gradually decreased from 28 in the first set to 10 in the second set and to 6 ± 1 in the

fifth set, for a total of 57 repetitions. This clearly suggests a flatter slope in the decline of

contractile work output after the first sets, particularly from sets 3 to 5 where the slope is

approaching a plateau. Similar results can be found in yet other studies involving multiple

sets of BFRRE performed to failure such as Nielsen et al16 and Farup et al8.

Based on this observed plateau combined with the knowledge of the fatigue and

recovery of the differerent types of muscle fibres, the following modification to the scenario

of Wernbom et al19 is suggested: in a series of 4-5 sets of low-load BFRRE to failure, type I

fibres become highly fatigued during the first set, thus necessitating the recruitment of type II

fibres. However, type II fibres will inevitably also become fatigued. Because the plateau is

largely reached already after the second set, it is suggested that type II fibres also are severely

fatigued at this point. In result and since type I fibres have a superior rate of recovery in both

metabolites and force, they will produce the majority of the force output from set 3 and

onwards. The maintained partial occlusion pressure will if anything probably put the type II

fibres at a further disadvantage, and the type IIX and IIAX fibres will soon become

ineffective due to the lack of recovery and may even drop out when approaching contraction

failure.

This article is protected by copyright. All rights reserved.


A lower force production in the muscle fibres would be predicted to result in

reduced glycogen use, as glycogenolysis and glycolysis are related to the ATP turnover in the
Accepted Article
contracting muscle, which in turn is related to the force development166,365. The scenario of

markedly decreased forces and lower glycogenolytic rates in type II fibres after the first two

sets of BFRRE to failure, (and even a drop-out of some fibres) would thus largely explain the

findings of Cumming et al21 of less glycogen depletion in the type II fibres than in type I

fibres. Conversely, however, the ischemic conditions will accelerate the glycogenolysis in the

type I fibres as previously discussed, and the type I fibres will presumably recover their force

development sufficiently between exercise sets for a relatively high rate of glycogenolysis in

this fibre type to be maintained. Most importantly, the type I fibres will be exposed to a far

greater number of contractions in total.

In low-load BFRRE protocols of 4-5 sets with fixed number of repetitions (e.g.

30-15-15-15), motor unit recruitment typically will be submaximal in the first two sets,

whereas in the last sets the effort may well become near-maximal. Assuming a largely intact

size principle for motor unit recruitment, it is suggested that it is mainly in the later sets of

high efforts that type IIAX and type IIX fibres will be engaged to a significant degree. Still,

type I fibres will likely be recruited for a far greater number of contractions.

The scenarios outlined above mainly concern unilateral BFR resistance training,

engaging a relatively small total muscle mass (ranging from e.g. wrist and elbow flexors to

the quadriceps). As outlined in Section 3.7., performing bilateral exercises will increase the

magnitude of group III/IV afferent inflow to the CNS, and likely inhibit the maximun muscle

activation levels reached during severely fatiguing exercise. This will almost certainly apply

to BFRRE as well. Consequently, it may be hypothesised that performing bilateral knee

extensions with occlusion can result in lower peak muscle activation levels than unilateral

knee extensions with occlusion.

This article is protected by copyright. All rights reserved.


In our experience, it is very demanding even for well-trained athletes to perform

bilateral multijoint movements such as squats and leg presses at very low loads to failure with
Accepted Article
BFR. Accordingly, it is postulated that the voluntary drive during large muscle mass

exercises with BFR at ~20-30% of 1RM may rarely reach levels sufficiently high to recruit

the highest threshold motor units and their muscle fibres. Some support for this notion may

be found in the study of Bjørnsen et al23, who in high-level powerlifters observed markedly

lower EMG amplitudes during low-load bilateral front squats performed to failure with BFR

as compared with bilateral front squats with heavy resistance but without BFR.

However, as mentioned earlier, it is also possible that some fibres may

experience acute and severe reductions in excitability and force production and even drop out

during such protocols, along with decreased motor unit firing rates, all of which could

contribute to the attenuated EMG amplitudes observed in the low-load BFRRE protocol.

Such factors could conceivably act in concert with submaximal recruitment and too short

inter-set recovery periods to limit the training-induced hypertrophic effects in type II fibres,

as elaborated below.

7.3. Consequences for hypertrophic adaptations to BFRRE

As noted above, low-load BFRRE to failure will initially cause severe fatigue in the type I

fibres in the first 1-2 sets. Especially towards the end of the first sets, type II fibres will

probably be primarily responsible for the force production in the working muscle, since the

severely fatigued type I fibres by then will produce relatively little force. Consequently, the

type II fibres may be exposed to relatively high levels of tension development, as previously

suggested307. An illustrative analogy would be various models of compensatory overload,

where synergist muscles are rendered inactive by surgical procedures (tenotomy, synergist

removal or denervation) leaving the remaining muscle(s) having to compensate for the loss of

This article is protected by copyright. All rights reserved.


active muscle, which induces muscle hypertrophy366. BFRRE could thus be seen as a

temporary functional ablation of the type I fibres as they become fatigued, leaving type II
Accepted Article
fibres to be mainly responsible for generating contractile force.

After the first 1-2 sets, fatigue will result in markedly lower force development

in type II fibres. At the same time, the greater recovery capacity of the type I fibres and the

shorter duration of the subsequent sets will shift more and more of the relative load back to

the type I fibres. As a result, type I fibres likely will be exposed to a greater time-tension

integral, and perhaps experience an overall stronger stimulation of hypertrophy signaling

through mechanotransduction pathways, than type II fibres. The large HSP responses in type

I fibres observed in the study of Cumming et al21 support this scenario, although possibly also

resulting from synergistic interactions between mechanical and ischemic stresses.

The presence of additive or synergistic interaction(s) between the mechanical

and ischemic stimuli in BFRRE has been hypothesised previously5,367 and might in part

explain how loads as low as 20% of 1RM can result in marked muscle hypertrophy, despite

seemingly producing only moderate mechanical stress even in the ”functional ablation”

scenario outlined above. Although still somewhat speculative, there are good reasons to

postulate such interactions, given that heat stress alone and repeated brief ischemia-

reperfusion alone have been shown to induce muscle hypertrophy in some studies368,369 and

that concomitant blood flow restriction can potentiate the muscle hypertrophy370-372 and

strength gains370,373 resulting from electrically stimulated contractions. Furthermore, Goto et

al374 reported that unilateral low-intensity resistance exercise for the elbow flexors combined

with heat stress in the same arm resulted in increased elbow flexor muscle CSA, whereas the

same unilateral exercise protocol alone did not produce any hypertrophy in the contralateral

arm.

This article is protected by copyright. All rights reserved.


However, if mechanical stimuli are important for the effects of BFRRE, the

typical way of performing BFRRE, i.e. 3-5 sets of high-number repetitions with short inter-
Accepted Article
set rest periods and with maintained partial occlusion, may not necessarily represent the

optimal choice for eliciting type II fibre hypertrophy and type II fibre-related strength and

power gains. In the case of multiple sets performed to failure, the number of repetitions with

relatively high tension in these fibres will probably be limited for reasons discussed above. It

may be speculated that once active force generation is markedly reduced in the muscle fibres

due to severe fatigue, this will limit any further synergistic effects between

mechanotransduction and ischemic stresses on hypertrophy signaling, particularly in type II

fibres. In the case of multiple sets of standardized repetitions (e.g. 30-15-15-15), the last 1-2

sets are generally the ones with the highest muscle activation and likely favoring the most

extensive type II fibre recruitment. Here too, the number of repetitions performed with

relatively high tension development in type II fibres might be limited.

Nevertheless, a relatively low volume of repetitions with high tension

development in the type II fibres during BFRRE is not necessarily problematic, as already

~10 repetitions in total in conventional heavy resistance training can result in measurable

muscle hypertrophy (reviewed by Wernbom et al375) accompanied by both type I and type II

myofibre hypertrophy376. Furthermore, the results of Nielsen et al16 demonstrate that

substantial hypertrophy in both type I and type II fibres can be achieved in a short time period

with high-frequency (once to twice daily) unilateral BFRRE. In contrast, a recent study by

Bjørnsen et al24, who employed greater volumes of BFRRE per session than Nielsen et al16,

showed temporary decreases (particularly in type II fibres) and subsequent delayed increases

in myofibre areas after short-term high-frequency BFRRE, accompanied by delayed gains in

muscle strength. Interestingly, the temporary type II atrophy in the study of Bjørnsen et al24

This article is protected by copyright. All rights reserved.


was correlated with the increased mRNA expression of p21, which is upregulated in skeletal

muscle during various atrophic conditions (see Bjørnsen et al24 for discussion).
Accepted Article
Thus, there seems to be a relatively narrow window of overall training stress per

session that is well tolerated by the muscles, and especially so for high-frequency BFRRE,

and that exceeding a certain threshold can be detrimental for the resulting muscle adaptations.

This delicate balance may in part explain why exercising to volitional failure in low-load

BFRRE may not necessarily be more effective for inducing gains in muscle strength and size

than BFRRE with submaximal effort, even at more normal training frequencies377.

Furthermore, there is evidence that high volumes of BFR exercise may be

unproductive in terms of muscle strength even in the absence of measurable muscle damage

and stress. For example, Jakobsgaard et al22 did not find any increases in myofiber areas or

muscle strength after a 6-week training period with three weekly sessions of high-repetition

bodyweight squats with BFR, despite modest but statistically significant gains in quadriceps

CSA, myonuclear addition in type I fibres, and considerable increases in endurance capacity

(ending up at ~200-300 repetitions per session). We therefore propose that BFR exercise

protocols involving several hundreds of repetitions in total may simply be too endurance-

oriented to induce muscle adaptations that result in marked increases in maximal muscle

strength and power. In light of the pronounced upregulation of p21 mRNA that has been

found after strenuous BFRRE (discussed in Bjørnsen et al24), there may also have been

insufficient recovery between the high-volume training sessions in Jakobsgaard et al22 for

optimal muscle adaptations to take place.

Finally, differences in training frequency and the use of unilateral single joint

low-load BFRRE in Nielsen et al16 as opposed to bilateral multi-joint low-load BFRRE22,23

may also have contributed to the contrasting results of these studies with reference to

preferential type I vs type II fibre hypertrophy and myonuclear addition. Thus, submaximal

This article is protected by copyright. All rights reserved.


recruitment and firing rates of high-threshold motorneurons innervating type II fibers due to

the especially pronounced inhibitory influence of group III/IV afferents in large muscle mass
Accepted Article
exercises, and an overall too low force-time integral in these myofibres during the exercise

may in part explain the hypertrophy in type I myofibres but no change in type II fibre area in

powerlifters in response to a 7-wk training cycle that incorporated low-load BFRRE23.

7.4. Consequences for neural adaptations to BFRRE

It is suggested that because of the inhibitory influence of group III/IV afferent input to the

CNS and the pronounced spike-frequency adaptation during ischemic exercise, a limit might

be imposed on the firing rates in the highest-threshold units. It is thus possible that such units

will not reach near-maximal discharge frequencies during a typical high-repetition BFRRE

protocol even during the repetitions with the overall highest activation, much like the

situation in slowly ramped-up isometric MVCs (cf. Section 3.6). This may be particularly

true when large muscle mass exercises are combined with BFR. In turn, this would result in

less-than-optimal neural adaptations from a strength and power perspective.

In support of this notion, several studies have reported that low-load BFRRE induces

strength gains and muscle hypertrophy without any significant changes in measures of

voluntary activation and neural drive378-380. In addition, high load strength training appears to

induce greater neural adaptations than low-load BFRRE378,380. Combined, this information

indicates that strategies and training programs that are designed to enhance the degree of

neuromuscular activation during BFRRE are warranted if the goal is to optimize neural

adaptations.

This article is protected by copyright. All rights reserved.


7.5. Consequences for interpretations of surface EMG during BFRRE

To summarize from Section 6.4.1., it is hypothesised that the seemingly submaximal EMG
Accepted Article
levels during very low load BFRRE, which are seen even at very high levels of effort are in

large part due to the combined effects of decreases in motor unit firing rates and attenuated

muscle fibre excitability, potentially compounded by neuromuscular transmission failure in

some fibres. Since any combination of these three factors can result in decrements in EMG

activity, it may also be argued that using surface EMG recording to assess muscle activation

levels at the start and at peak exercise may underestimate the number of muscle fibres that

have been activated during a BFRRE protocol. For example, an EMG RMS amplitude during

low-load BFRRE which reaches only 60% of MVC even at concentric failure does not

necessarily exclude that the highest threshold units (innervating type IIX and/or IIAX fibres)

are recruited, as some of the motor units may demonstrate lowered firing rates and/or a

reduced number of active muscle fibres at this point.

As the muscle mass involved increases from unilateral to bilateral single joint to

bilateral multi-joint exercises, the influence of central fatigue (largely mediated by group

III/IV afferent input) also is expected to increase, which will probably affect both recruitment

thresholds and firing rates of motor units negatively during fatiguing low-load BFRRE.

Hypothetically, this will in turn be reflected in lower maximal EMG amplitudes during low-

load BFRRE with bilateral multi-joint exercise compared with unilateral single joint exercise.

On the other hand, since sEMG may not detect the deepest motor units and thus

is inherently limited to the more superficial parts of muscles, and because several muscles

and muscle groups display a more or less pronounced deep-to-superficial activation pattern, it

is suggested that fatigue and neuromuscular transmission failure can develop in the deeper

parts of the involved muscles during very strenuous low-load BFRRE without this being

readily detected in the sEMG recordings.

This article is protected by copyright. All rights reserved.


Given the similarities between BFRRE and low-load training to fatigue without

BFR8,19,314,333, and that some degree of intramuscular blood flow restriction can occur during
Accepted Article
free-flow (i.e. non-occluded ) resistance exercise already at relatively low loading intensities

(reviewed previously by Wernbom et al5), many points discussed in this review may to

varying extent apply also to low-to-moderate load resistance exercise performed to fatigue

without using concurrent external occlusion.

7.6. Consequences for the possible role of metabolic stress in BFRRE

Summarized from Section 6.5., it appears possible that in strenuous low-load ischemic

exercise, Pi splits may occur not only due to activation of type IIA fibres in addition to the

already active but fatiguing type I fibres, but also largely as a result of subsequent progressive

recruitment of type IIAX and/or type IIX fibres due to accumulating fatigue in type I and type

IIA fibres. In addition, non-localized 31P-MRS measurements must be viewed with caution,

as it cannot be ruled out that Pi splits are at least in part caused by signal contrasts between

active and less active or even inactive muscles.

Importantly, these and other uncertainties in the literature on 31P-MRS

measurements during BFRRE would seem to seriously limit the utility of 31P-MRS as a

stand-alone method to probe the mechanisms underpinning gains in muscle strength and

hypertrophy with this mode of exercise. Specifically, the strong correlations between acute

increases in Pi and training-induced gains in muscle area and strength that were reported by

Takada et al10 are open to question. Given that Pi increases concomittantly with decreases in

PCr158, and that gradually increasing fatigue is a major driver of the successive recruitment of

new motor units in low-load BFRRE, a greater elevation of Pi is likely to result not only from

more extensive depletion of PCr in each active myofibre (reflecting increased metabolic

stress) but also to stem from recruitment of a larger number of muscle fibres.

This article is protected by copyright. All rights reserved.


Therefore, the correlations between acute elevations in Pi and longer-term

increases in muscle CSA and strength in Takada et al10 may actually reflect the importance of
Accepted Article
activating a large number of muscle fibres (and thereby exposing them to significant

mechanical stress) to cause these gains, as opposed to a role of metabolic stress accumulation.

In other words, the relationships reported by Takada et al10 do not constitute firm evidence of

a role for metabolic stress per se in BFRRE, as the correlations may be partly or even fully

coincidental. However, we propose that the most likely scenario is a synergistic or additive

interaction between mechanical stimuli and metabolic stress and/or other types of ischemic

stimuli, as elaborated in detail above.

Acknowledgements

The authors gratefully thank Dr Kristoffer Cumming for valuable help with the pictures on

PAS staining of muscle fibres. Mathias Wernbom gratefully acknowledges the The Swedish

Research Council for Sport Science for post-doctoral fundings during the years 2013 to 2016,

and for supporting several research projects on BFRRE during the years 2004 to 2009.

Funding

No grants or funds were received to support this work.

Conflict of Interest

The authors declare no conflict of interest.

This article is protected by copyright. All rights reserved.


References

1. Takarada Y, Takazawa H, Sato Y, et al. Effects of resistance exercise combined with moderate
Accepted Article
vascular occlusion on muscular function in humans. J Appl Physiol. 2000: 88: 2097-2106.
2. Laurentino GC, Ugrinowitsch C, Roschel H, et al. Strength training with blood flow restriction
diminishes myostatin gene expression. Med Sci Sports Exerc. 2012; 44:406-412.
3. Ellefsen S, Hammarström D, Strand TA, et al. Blood flow-restricted strength training displays
high functional and biological efficacy in women: a within-subject comparison with high-load
strength training. Am J Physiol Regul Integr Comp Physiol. 2015;309:R767-779.
4. Lixandrão ME, Ugrinowitsch C, Berton R, et al. Magnitude of Muscle Strength and Mass
Adaptations Between High-Load Resistance Training Versus Low-Load Resistance Training
Associated with Blood-Flow Restriction: A Systematic Review and Meta-Analysis. Sports
Med. 2018;48:361-378.
5. Wernbom M, Augustsson J, Raastad T. Ischemic strength training: a low-load alternative to
heavy resistance exercise? Scand J Med Sci Sports. 2008; 18: 401-16.
6. Hughes L, Paton B, Rosenblatt B, et al. Blood flow restriction training in clinical
musculoskeletal rehabilitation: a systematic review and meta-analysis. Br J Sports Med.
2017;51:1003-1011.
7. Abe T, Loenneke JP, Fahs CA, et al. Exercise intensity and muscle hypertrophy in blood flow-
restricted limbs and non-restricted muscles: a brief review. Clin Physiol Funct Imaging.
2012;32:247-252.
8. Farup J, de Paoli F, Bjerg K, et al. Blood flow restricted and traditional resistance training
performed to fatigue produce equal muscle hypertrophy. Scand J Med Sci Sports.
2015;25:754-763.
9. Suga T, Okita K, Takada S, et al. Effect of multiple set on intramuscular metabolic stress
during low-intensity resistance exercise with blood flow restriction. Eur J Appl Physiol. 2012;
112: 3915-20.
10. Takada S, Okita K, Suga T, et al. Low-intensity exercise can increase muscle mass and
strength proportionally to enhanced metabolic stress under ischemic conditions. J Appl
Physiol. 2012;113:199-205.
11. Aagaard P, Andersen JL, Dyhre-Poulsen P, et al. A mechanism for increased contractile
strength of human pennate muscle in response to strength training: changes in muscle
architecture. J Physiol. 2001;534:613-623.
12. Kosek DJ, Kim JS, Petrella JK, et al. Efficacy of 3 days/wk resistance training on myofiber
hypertrophy and myogenic mechanisms in young vs. older adults. J Appl Physiol.
2006;101:531-544.
13. Suetta C, Andersen JL, Dalgas U, et al. Resistance training induces qualitative changes in
muscle morphology, muscle architecture, and muscle function in elderly postoperative
patients. J Appl Physiol. 2008;105:180-186.
14. Jakobsen MD, Sundstrup E, Randers MB, et al. The effect of strength training, recreational
soccer and running exercise on stretch-shortening cycle muscle performance during
countermovement jumping. Hum Mov Sci. 2012;31:970-986.
15. Faulkner JA, Claflin DR, McCully KK. Power output of fast and slow fibers from human
skeletal muscle. In: Human Muscle Power, edited by Jones NL, McCartney N and McComas
AJ, Champaign Illinois, Human Kinetics 1986, pp 81-94.
16. Nielsen JL, Aagaard P, Bech RD, et al. Proliferation of myogenic stem cells in human skeletal
muscle in response to low-load resistance training with blood flow restriction. J Physiol.
2012;590:4351-4361.
17. Moritani T, Sherman WM, Shibata M, et al. Oxygen availability and motor unit activity in
humans. Eur J Appl Physiol 1992;64:552-556.

This article is protected by copyright. All rights reserved.


18. Takarada Y, Nakamura Y, Aruga S, et al. Rapid increase in plasma growth hormone after low-
intensity resistance exercise with vascular occlusion. J Appl Physiol. 2000;88:61-65.
19. Wernbom M, Järrebring R, Andreasson MA, Augustsson J. Acute effects of blood flow
restriction on muscle activity and endurance during fatiguing dynamic knee extensions at
Accepted Article
low load. J Strength Cond Res. 2009;23:2389-2395.
20. Krustrup P, Söderlund K, Relu MU, et al. Heterogeneous recruitment of quadriceps muscle
portions and fibre types during moderate intensity knee-extensor exercise: effect of thigh
occlusion. Scand J Med Sci Sports. 2009;19:576-584.
21. Cumming KT, Paulsen G, Wernbom M, et al. Acute response and subcellular movement of
HSP27, αB-crystallin and HSP70 in human skeletal muscle after blood-flow-restricted low-
load resistance exercise. Acta Physiol. 2014; 211:634-646.
22. Jakobsgaard JE, Christiansen M, Sieljacks P, et al K. Impact of blood flow-restricted
bodyweight exercise on skeletal muscle adaptations. Clin Physiol Funct Imaging. 2018 Feb
15. doi: 10.1111/cpf.12509. [Epub ahead of print]
23. Bjørnsen T, Wernbom M, Kirketeig A, et al. Type 1 muscle fiber hypertrophy after blood
flow-restricted training in powerlifters. Med Sci Sports Exerc. 2019;51:288-298.
24. Bjørnsen T, Wernbom M, Løvstad AT, et al. Delayed myonuclear addition, myofiber
hypertrophy and increases in strength with high-frequency low-load blood flow restricted
training to volitional failure. J Appl Physiol. 2019;126:578-592.
25. Sumide T, Sakuraba K, Sawaki K, Ohmura H, Tamura Y. Effect of resistance exercise training
combined with relatively low vascular occlusion. J Sci Med Sport. 2009;12:107-112.
26. Shinohara M, Kouzaki M, Yoshihisa T, Fukunaga T. Efficacy of tourniquet ischemia for
strength training with low resistance. Eur J Appl Physiol. 1998;77:189-191.
27. Moritani T. Motor unit and motoneurone excitability during explosive movement. In:
Strength and Power in Sport, 2nd ed., edited by Paavo Komi, Oxford, Blackwell Science,
2003, pp 27-49.
28. Duchateau J, Semmler JG, Enoka RM. Training adaptations in the behavior of human motor
units. J Appl Physiol. 2006;101:1766-1775.
29. Chalmers GR. Can fast-twitch muscle fibres be selectively recruited during lengthening
contractions? Review and applications to sport movements. Sports Biomech. 2008;7:137-
157.
30. Heckman CJ, Enoka RM. Motor unit. Compr Physiol. 2012;2:2629-2682.
31. Burke RE, Levine DN, Zajac FE 3rd. Mammalian motor units: physiological-histochemical
correlation in three types in cat gastrocnemius. Science. 1971;174(4010):709-12.
32. Burke RE, Levine DN, Tsairis P, Zajac FE 3rd. Physiological types and histochemical profiles in
motor units of the cat gastrocnemius. J Physiol. 1973;234:723-748.
33. Edström L, Grimby L. Effect of exercise on the motor unit. Muscle Nerve. 1986;9:104-126.
34. McDonagh JC, Binder MD, Reinking RM, Stuart DG. Tetrapartite classification of motor units
of cat tibialis posterior. J Neurophysiol. 1980;44:696-712.
35. Burke RE. The structure and function of motor units. In: Disorders of Voluntary Muscle, 7th
ed., edited by Karpati G, Hilton D, Griggs RC, Cambridge University Press, 2001, pp 3-21.
36. Burke RE. Some unresolved issues in motor unit research. In: Sensorimotor Control of
Movement and Posture, edited by Gandevia S, Proske U, Stuart DG, Kluwer
Academic/Plenum Press, 2002, pp 171-178.
37. Garnett RA, O'Donovan MJ, Stephens JA, Taylor A. Motor unit organization of human medial
gastrocnemius. J Physiol. 1979;287:33-43.
38. Buchthal F, Schmalbruch H. Contraction times and fibre types in intact human muscle. Acta
Physiol Scand. 1970;79:435-452.
39. Ennion S, Sant'ana Pereira J, Sargeant AJ, et al. Characterization of human skeletal muscle
fibres according to the myosin heavy chains they express. J Muscle Res Cell Motil.
1995;16:35-43.

This article is protected by copyright. All rights reserved.


40. Sant'Ana Pereira JA, Sargeant AJ, Rademaker AC, et al. Myosin heavy chain isoform
expression and high energy phosphate content in human muscle fibres at rest and post-
exercise. J Physiol. 1996;496:583-588.
41. Staron RS, Herman JR, Schuenke MD. Misclassification of hybrid fast fibers in resistance-
Accepted Article
trained human skeletal muscle using histochemical and immunohistochemical methods. J
Strength Cond Res. 2012;26:2616-2622.
42. Sale DG. Influence of exercise and training on motor unit activation. Exerc Sport Sci Rev.
1987;15:95-151.
43. MacIntosh BR, Gardiner PF, McComas AJ. Skeletal Muscle: Form and Function, 2nd editon.
Champaign, Human Kinetics, 2006, pp 195-207.
44. Staron RS. Human skeletal muscle fiber types: delineation, development, and distribution.
Can J Appl Physiol. 1997;22:307-27.
45. Andersen JL, Klitgaard H, Saltin B. Myosin heavy chain isoforms in single fibres from m.
vastus lateralis of sprinters: influence of training. Acta Physiol Scand. 1994;151:135-142.
46. Andersen JL, Aagaard P. Myosin heavy chain IIX overshoot in human skeletal muscle. Muscle
Nerve. 2000;23:1095-1104.
47. Klitgaard H, Zhou M, Richter EA. Myosin heavy chain composition of single fibres from m.
biceps brachii of male body builders. Acta Physiol Scand. 1990;140:175-180.
48. Sant'ana Pereira JA, Wessels A, Nijtmans L, et al. New method for the accurate
characterization of single human skeletal muscle fibres demonstrates a relation between
mATPase and MyHC expression in pure and hybrid fibre types. J Muscle Res Cell Motil.
1995;16:21-34.
49. Karatzaferi C, de Haan A, Ferguson RA, et al. Phosphocreatine and ATP content in human
single muscle fibres before and after maximum dynamic exercise. Pflügers Arch.
2001;442:467-474.
50. Prats C, Gomez-Cabello A, Nordby P, et al. An optimized histochemical method to assess
skeletal muscle glycogen and lipid stores reveals two metabolically distinct populations of
type I muscle fibers. PLoS One. 2013 30;8(10):e77774.
51. Essén B, Jansson E, Henriksson J, et al. Metabolic characteristics of fibre types in human
skeletal muscle. Acta Physiol Scand. 1975;95:153-65.
52. Greenhaff PL, Söderlund K, Ren JM, Hultman E. Energy metabolism in single human muscle
fibres during intermittent contraction with occluded circulation. J Physiol. 1993;460:443-53.
53. Greenhaff PL, Nevill ME, Soderlund K, et al. The metabolic responses of human type I and II
muscle fibres during maximal treadmill sprinting. J Physiol. 1994;478:149-55.
54. Henneman E. Relation between size of neurons and their susceptibility to discharge. Science.
1957;126:1345-1347.
55. Henneman E, Somjen G, Carpenter DO. Functional significance of cell size in spinal
motoneurons. J Neurophysiol. 1965;28:560-580.
56. Milner-Brown HS, Stein RB, Yemm R. The orderly recruitment of human motor units during
voluntary isometric contractions. J Physiol. 1973; 230:359-370.
57. Freund HJ, Büdingen HJ, Dietz V. Activity of single motor units from human forearm muscles
during voluntary isometric contractions. J Neurophysiol. 1975;38:933-46.
58. Monster AW, Chan H. Isometric force production by motor units of extensor digitorum
communis muscle in man. J Neurophysiol. 1977;40:1432-43.
59. Stephens JA, Usherwood TP. The mechanical properties of human motor units with special
reference to their fatiguability and recruitment threshold. Brain Res. 1977;125:91-97.
60. Desmedt JE, Godaux E. Ballistic contractions in man: characteristic recruitment pattern of
single motor units of the tibialis anterior muscle. J Physiol. 1977;264:673-693.
61. Garnett R, Stephens JA. Changes in the recruitment threshold of motor units produced by
cutaneous stimulation in man. J Physiol. 1981;311:463-473.

This article is protected by copyright. All rights reserved.


62. Calancie B, Bawa P. Recruitment order of motor units during the stretch reflex in man. Brain
Res. 1984;292:176-178.
63. Bawa P, Lemon RN. Recruitment of motor units in response to transcranial magnetic
stimulation in man. J Physiol. 1993;471:445-464.
Accepted Article
64. Enoka RM, Fuglevand AJ. Motor unit physiology: some unresolved issues. Muscle Nerve.
2001;24:4-17.
65. Hannerz J. Discharge properties of motor units in relation to recruitment order in voluntary
contraction. Acta Physiol Scand. 1974;91:374-385.
66. Kukulka CG, Clamann HP. Comparison of the recruitment and discharge properties of motor
units in human brachial biceps and adductor pollicis during isometric contractions. Brain Res.
1981;219:45-55.
67. Christie A, Greig Inglis J, Kamen G, Gabriel DA. Relationships between surface EMG variables
and motor unit firing rates. Eur J Appl Physiol. 2009;107:177-185.
68. Oya T, Riek S, Cresswell AG. Recruitment and rate coding organisation for soleus motor units
across entire range of voluntary isometric plantar flexions. J Physiol. 2009;587:4737-4748.
69. De Luca CJ, Hostage EC. Relationship between firing rate and recruitment threshold of
motoneurons in voluntary isometric contractions. J Neurophysiol. 2010;104:1034-1046.
70. Bawa PN, Jones KE, Stein RB. Assessment of size ordered recruitment. Front Hum Neurosci.
2014;8:532.
71. Grimby L, Hannerz J. Firing rate and recruitment order of toe extensor motor units in
different modes of voluntary conraction. J Physiol. 1977;264:865-879.
72. Nardone A, Romanò C, Schieppati M. Selective recruitment of high-threshold human motor
units during voluntary isotonic lengthening of active muscles. J Physiol. 1989;409:451-471.
73. Grimby L. Single motor unit discharge during voluntary contraction and locomotion. In:
Human Muscle Power, edited by Jones NL, McCartney N and McComas AJ, Champaign
Illinois, Human Kinetics 1986, pp 111-130.
74. Hodges PW, Tucker K. Moving differently in pain: a new theory to explain the adaptation to
pain. Pain. 2011;152(3 Suppl):S90-8.
75. Kanda K, Burke RE, Walmsley B. Differential control of fast and slow twitch motor units in
the decerebrate cat. Exp Brain Res. 1977;29:57-74.
76. Masakado Y, Kamen G, De Luca CJ. Effects of percutaneous stimulation on motor unit firing
behavior in man. Exp Brain Res. 1991;86:426-32.
77. Binder MD, Heckman CJ, Powers RK. The physiological control of motoneuron activity. In:
Handbook of physiology. Exercise: regulation and integration of multiple systems, edited by
Rowell LB and Shepherd JT, American Physiological Society 1996, 12, pp 1-53.
78. Cope TC, Clark BD. Are there important exceptions to the size principle of α-motoneurone
recruitment? In: Alpha and gamma motor systems, edited by Taylor A, Gladden MH, and
Durban R, Springer, Boston, MA, 1995, pp. 71-78.
79. Bawa P, Jones KE. Do lengthening contractions represent a case of reversal in recruitment
order? Prog Brain Res. 1999;123:215-220.
80. Tucker K, Butler J, Graven-Nielsen T, et al. Motor unit recruitment strategies are altered
during deep-tissue pain. J Neurosci. 2009;29:10820-10826.
81. Tucker KJ, Hodges PW. Motoneurone recruitment is altered with pain induced in non-
muscular tissue. Pain. 2009;141:151-155.
82. Edwards RH, Young A, Hosking GP, Jones DA. Human skeletal muscle function: description of
tests and normal values. Clin Sci Mol Med. 1977;52:283-290.
83. Hultman E, Sjöholm H, Jäderholm-Ek I, Krynicki J. Evaluation of methods for electrical
stimulation of human skeletal muscle in situ. Pflugers Arch. 1983;398:139-141.
84. Gregory CM, Dixon W, Bickel CS. Impact of varying pulse frequency and duration on muscle
torque production and fatigue. Muscle Nerve. 2007;35:504-509.

This article is protected by copyright. All rights reserved.


85. Eberstein A, Goodgold J. Slow and fast twitch fibers in human skeletal muscle. Am J Physiol.
1968;215:535-541.
86. Moulds RF, Young A, Jones DA, Edwards RH. A study of the contractility, biochemistry and
morphology of an isolated preparation of human skeletal muscle. Clin Sci Mol Med.
Accepted Article
1977;52:291-297.
87. Bigland-Ritchie B, Rice CL, Garland SJ, Walsh ML. Task-dependent factors in fatigue of human
voluntary contractions. In: Fatigue. Edited by Gandevia SC, Enoka RM, McComas AJ et al.,
Springer, Boston, MA, 1995. pp. 361-380.
88. Desmedt JE, Godaux E. Ballistic contractions in fast or slow human muscles: discharge
patterns of single motor units. J Physiol. 1978;285:185-196.
89. Van Cutsem M, Duchateau J, Hainaut K. Changes in single motor unit behaviour contribute
to the increase in contraction speed after dynamic training in humans. J Physiol.
1998;513:295-305.
90. Abbate F, Bruton JD, De Haan A, Westerblad H. Prolonged force increase following a high-
frequency burst is not due to a sustained elevation of [Ca2+]i. Am J Physiol Cell Physiol.
2002;283:C42-47.
91. Cheng AJ, Place N, Bruton JD, et al. Doublet discharge stimulation increases sarcoplasmic
reticulum Ca2+ release and improves performance during fatiguing contractions in mouse
muscle fibres. J Physiol. 2013;591:3739-3748.
92. Kamen G, Knight CA. Training-related adaptations in motor unit discharge rate in young and
older adults. J Gerontol A Biol Sci Med Sci. 2004;59:1334-1338.
93. Christie A, Kamen G. Short-term training adaptations in maximal motor unit firing rates and
afterhyperpolarization duration.Muscle Nerve. 2010;41:651-660.
94. Knight CA, Kamen G. Relationships between voluntary activation and motor unit firing rate
during maximal voluntary contractions in young and older adults. Eur J Appl Physiol.
2008;103:625-630.
95. Woods JJ, Furbush F, Bigland-Ritchie B. Evidence for a fatigue-induced reflex inhibition of
motoneuron firing rates. J Neurophysiol. 1987;58:125-137.
96. Strojnik V. Muscle activation level during maximal voluntary effort. Eur J Appl Physiol Occup
Physiol. 1995;72:144-149.
97. Behm DG, Whittle J, Button D, Power K. Intermuscle differences in activation. Muscle Nerve.
2002;25:236-243.
98. Babault N, Pousson M, Michaut A, Ballay Y, Hoecke JV. EMG activity and voluntary activation
during knee-extensor concentric torque generation. Eur J Appl Physiol. 2002;86:541-547.
99. Beltman JG, Sargeant AJ, van Mechelen W, de Haan A. Voluntary activation level and muscle
fiber recruitment of human quadriceps during lengthening contractions. J Appl Physiol.
2004;97:619-626.
100. Place N, Casartelli N, Glatthorn JF, Maffiuletti NA. Comparison of quadriceps
inactivation between nerve and muscle stimulation. Muscle Nerve. 2010;42:894-900.
101. Van Leeuwen DM, De Ruiter CJ, De Haan A. Effect of stimulation intensity on
assessment of voluntary activation. Muscle Nerve. 2012;45:841-848.
102. Pucci AR, Griffin L, Cafarelli E. Maximal motor unit firing rates during isometric
resistance training in men. Exp Physiol. 2006;91:171-178.
103. Grimby L, Hannerz J, Hedman B. Contraction time and voluntary discharge properties
of individual short toe extensor motor units in man. J Physiol. 1979; 289:191-201.
104. Bellemare F, Woods JJ, Johansson R, Bigland-Ritchie B. Motor-unit discharge rates in
maximal voluntary contractions of three human muscles. J Neurophysiol. 1983;50:1380-
1392.
105. Grimby L, Hannerz J, Hedman B. The fatigue and voluntary discharge properties of
single motor units in man. J Physiol. 1981;316:545-554.

This article is protected by copyright. All rights reserved.


106. Kanosue K, Yoshida M, Akazawa K, Fujii K. The number of active motor units and their
firing rates in voluntary contraction of human brachialis muscle. Jpn J Physiol. 1979;29:427-
443.
107. Harwood B, Davidson AW, Rice CL. Motor unit discharge rates of the anconeus muscle
Accepted Article
during high-velocity elbow extensions. Exp Brain Res. 2011;208:103-113.
108. Hannerz J, Grimby L. The afferent influence on the voluntary firing range of individual
motor units in man. Muscle Nerve. 1979;2:414-422.
109. Gydikov A, Kosarov D. Some features of different motor units in human biceps brachii.
Pflugers Arch. 1974;347:75-88.
110. De Luca CJ, LeFever RS, McCue MP, Xenakis AP. Behaviour of human motor units in
different muscles during linearly varying contractions. J Physiol. 1982;329:113-128.
111. De Luca CJ, Erim Z. Common drive of motor units in regulation of muscle force. Trends
Neurosci. 1994;17:299-305.
112. Erim Z, De Luca CJ, Mineo K, Aoki T. Rank-ordered regulation of motor units. Muscle
Nerve. 1996;19:563-573.
113. De Luca CJ, Contessa P. Hierarchical control of motor units in voluntary contractions. J
Neurophysiol. 2012;107:178-195.
114. Person RS, Kudina LP. Discharge frequency and discharge pattern of human motor
units during voluntary contraction of muscle. Electroencephalogr Clin Neurophysiol.
1972;32:471-483.
115. Tanji J, Kato M. Recruitment of motor units in voluntary contraction of a finger muscle
in man. Exp Neurol. 1973;40:759-770.
116. Enoka RM, Duchateau J. Rate coding and the control of muscle force. Cold Spring Harb
Perspect Med. 2017;7(10): pii: a029702.
117. Piotrkiewicz M, Türker K. Onion skin or common drive? Front Cell Neurosci 2017;11:2.
118. Kosarov D, Gydikov A. Dependence of the discharge frequency of motor units in
different human muscles upon the level of the isometric muscle tension. Electromyogr Clin
Neurophysiol. 1976;16:293-306.
119. Moritz CT, Barry BK, Pascoe MA, Enoka RM. Discharge rate variability influences the
variation in force fluctuations across the working range of a hand muscle. J Neurophysiol.
2005;93:2449-2459.
120. Barry BK, Pascoe MA, Jesunathadas M, Enoka RM. Rate coding is compressed but
variability is unaltered for motor units in a hand muscle of old adults. J Neurophysiol.
2007;97:3206-3218.
121. Grimby L, Hannerz J. Recruitment order of motor units on voluntary contraction:
changes induced by proprioceptive afferent activity. J Neurol Neurosurg Psychiatry.
1968;31:565-573.
122. Gatev P, Ivanova T, Gantchev GN. Changes in the firing pattern of high-threshold
motor units due to fatigue. Electromyogr Clin Neurophysiol. 1986;26:83-93.
123. Warmolts JR, Engel WK. Open-biopsy electromyography. I. Correlation of motor unit
behavior with histochemical muscle fiber type in human limb muscle. Arch Neurol.
1972;27:512-517.
124. Warmolts JR, Engel WK. Correlation of motor unit behavior with histochemical
myofiber type in humans by open-biopsy electromyography. In: New Concepts of the Motor
Unit, Neuromuscular Disorders, Electromyographic Kinesiology. Karger Publishers, 1973. p.
35-40.
125. Grimby L. Firing properties of single human motor units during locomotion. J Physiol.
1984;346:195-202.
126. Søgaard K, Christensen H, Fallentin N, et al. Motor unit activation patterns during
concentric wrist flexion in humans with different muscle fibre composition. Eur J Appl Physiol
Occup Physiol. 1998;78:411-416.

This article is protected by copyright. All rights reserved.


127. Grimby L, Hannerz J, Borg J, Hedman B. Firing properties of single human motor units
on maintained maximal voluntary effort. In: Human Muscle Fatigue: Physiological
Mechanisms. Chichester, UK: John Wiley & Sons, Ltd., 1981. pp 157-177.
128. Van Cutsem M, Duchateau J. Preceding muscle activity influences motor unit
Accepted Article
discharge and rate of torque development during ballistic contractions in humans. J Physiol.
2005;562:635-644.
129. Harwood B, Choi I, Rice CL. Reduced motor unit discharge rates of maximal velocity
dynamic contractions in response to a submaximal dynamic fatigue protocol. J Appl Physiol.
2012;113:1821-1830.
130. Sawczuk A, Powers RK, Binder MD. Intrinsic properties of motoneurons. In: Fatigue.
Springer, Boston, MA, 1995. p. 123-134.
131. Kernell D, Eerbeek O, Verhey BA. Relation between isometric force and stimulus rate
in cat's hindlimb motor units of different twitch contraction time. Exp Brain Res.
1983;50:220-227.
132. Zengel JE, Reid SA, Sypert GW, Munson JB. Membrane electrical properties and
prediction of motor-unit type of medial gastrocnemius motoneurons in the cat. J
Neurophysiol. 1985;53:1323-1344.
133. Gardiner PF. Physiological properties of motoneurons innervating different muscle
unit types in rat gastrocnemius. J Neurophysiol. 1993;69:1160-1170.
134. Bigland-Ritchie BR, Dawson NJ, Johansson RS, Lippold OC. Reflex origin for the slowing
of motoneurone firing rates in fatigue of human voluntary contractions. J Physiol.
1986;379:451-9.
135. Taylor JL, Amann M, Duchateau J, et al. Neural contributions to muscle fatigue: from
the brain to the muscle and back Again. Med Sci Sports Exerc. 2016;48:2294-2306.
136. Amann M, Sidhu SK, Weavil JC, et al. Autonomic responses to exercise: group III/IV
muscle afferents and fatigue. Auton Neurosci. 2015;188:19-23.
137. Windhorst U, Kirmayer D, Soibelman F, et al. Effects of neurochemically excited group
III-IV muscle afferents on motoneuron afterhyperpolarization. Neuroscience. 1997;76:915-
929.
138. Martin PG, Weerakkody N, Gandevia SC, Taylor JL. Group III and IV muscle afferents
differentially affect the motor cortex and motoneurones in humans. J Physiol.
2008;586:1277-1289.
139. Garland SJ, Griffin L, Ivanova T. Motor unit discharge rate is not associated with
muscle relaxation time in sustained submaximal contractions in humans. Neurosci Lett.
1997;239:25-28.
140. Bonde-Petersen F, Mork AL, Nielsen E. Local muscle blood flow and sustained
contractions of human arm and back muscles. Eur J Appl Physiol Occup Physiol. 1975;34:43-
50.
141. Griffin L, Garland SJ, Ivanova T, Hughson RL. Blood flow in the triceps brachii muscle in
humans during sustained submaximal isometric contractions. Eur J Appl Physiol.
2001;84:432-437.
142. Freund PR, Hobbs SF, Rowell LB. Cardiovascular responses to muscle ischemia in man–
dependency on muscle mass. J Appl Physiol Respir Environ Exerc Physiol. 1978;45:762-767.
143. Mitchell JH, Payne FC, Saltin B, Schibye B. The role of muscle mass in the
cardiovascular response to static contractions. J Physiol. 1980;309:45-54.
144. Seals DR. Influence of muscle mass on sympathetic neural activation during isometric
exercise. J Appl Physiol. 1989;67:1801-1806.
145. Seals DR. Influence of active muscle size on sympathetic nerve discharge during
isometric contractions in humans. J Appl Physiol. 1993;75:1426-1431.

This article is protected by copyright. All rights reserved.


146. Matkowski B, Place N, Martin A, Lepers R. Neuromuscular fatigue differs following
unilateral vs bilateral sustained submaximal contractions. Scand J Med Sci Sports.
2011;21:268-276.
147. Rossman MJ, Venturelli M, McDaniel J, et al. Muscle mass and peripheral fatigue: a
Accepted Article
potential role for afferent feedback? Acta Physiol. 2012;206:242-250.
148. Rossman MJ, Garten RS, Venturelli M, et al. The role of active muscle mass in
determining the magnitude of peripheral fatigue during dynamic exercise. Am J Physiol
Regul Integr Comp Physiol. 2014;306:R934-340.
149. Dietz V. Analysis of the electrical muscle activity during maximal contraction and the
influence of ischaemia. J Neurol Sci. 1978;37:187-197.
150. Carvalho AJ, McKee NH. Active and passive forces during ischemia and reperfusion of
rat fast and slow twitch skeletal muscles. Adv Exp Med Biol. 1992;311:365-368.
151. Carvalho AJ, McKee NH, Green HJ. Metabolic and contractile responses of fast- and
slow-twitch rat skeletal muscles to ischemia. Can J Physiol Pharmacol. 1996;74:1333-1341.
152. Gossen ER, Ivanova TD, Garland SJ. Ischemia sensitivity and motoneuron
afterhyperpolarization in human motor units. Muscle Nerve. 2004;30:195-201.
153. Folkow B, Halicka H. A comparison between “red” and “white” muscle with respect to
blood supply, capillary surface area and oxygen uptake during rest and exercise. Microvasc
Res. 1968;1:1-14.
154. Sahlin K, Edström L, Sjöholm H. Force, relaxation and energy metabolism of rat soleus
muscle during anaerobic contraction. Acta Physiol Scand. 1987;129:1-7.
155. Gollnick PD, Karlsson J, Piehl K, Saltin B. Selective glycogen depletion in skeletal
muscle fibres of man following sustained contractions. J Physiol. 1974;241:59-67.
156. Murthy G, Hargens AR, Lehman S, Rempel DM. Ischemia causes muscle fatigue. J
Orthop Res. 2001; 19:436-440.
157. Bylund-Fellenius AC, Walker PM, Elander A, et al. Energy metabolism in relation to
oxygen partial pressure in human skeletal muscle during exercise. Biochem J. 1981;200:247-
255.
158. Allen DG, Lamb GD, Westerblad H. Skeletal muscle fatigue: cellular mechanisms.
Physiol Rev. 2008;88:287-332.
159. Allen DG, Trajanovska S. The multiple roles of phosphate in muscle fatigue. Front
Physiol. 2012;3:463.
160. Karatzaferi C, Franks-Skiba K, Cooke R. Inhibition of shortening velocity of skinned
skeletal muscle fibers in conditions that mimic fatigue. Am J Physiol Regul Integr Comp
Physiol. 2008;294:R948-955.
161. Nelson CR, Debold EP, Fitts RH. Phosphate and acidosis act synergistically to depress
peak power in rat muscle fibers. Am J Physiol Cell Physiol. 2014;307:C939-950.
162. Debold EP. Decreased myofilament calcium sensitivity plays a significant role in
muscle fatigue. Exerc Sport Sci Rev. 2016;44:144-149.
163. Hultman E, Del Canale S, Sjöholm H. Effect of induced metabolic acidosis on
intracellular pH, buffer capacity and contraction force of human skeletal muscle. Clin Sci.
1985;69:505-510.
164. Hultman E, Sjöhom H, Sahlin K, Edström L. Glycolytic and oxidative energy metabolism
and contraction characteristics of intact human muscle. In: Human Muscle Fatigue:
Physiological Mechanisms. Chichester, UK: John Wiley & Sons, Ltd., 1981. pp 19-40.
165. Hultman E, Sjöholm H. Electromyogram, force and relaxation time during and after
continuous electrical stimulation of human skeletal muscle in situ. J Physiol. 1983;339:33-40.
166. Hultman E, Sjöholm H. Biochemical causes of fatigue. In: Human Muscle Power. Jones
NL, McCartney N, McComas AJ (Editors). Champaign, Illinois: Human Kinetics Publishers
1986, pp 215-238.

This article is protected by copyright. All rights reserved.


167. Söderlund K, Hultman E. ATP content in single fibres from human skeletal muscle after
electrical stimulation and during recovery. Acta Physiol Scand. 1990;139:459-466.
168. Söderlund K, Greenhaff PL, Hultman E. Energy metabolism in type I and type II human
muscle fibres during short term electrical stimulation at different frequencies. Acta
Accepted Article
Physiologica Scandinavica. 1992;144:15-22.
169. Söderlund K, Hultman E. ATP and phosphocreatine changes in single human muscle
fibers after intense electrical stimulation. Am J Physiol 1991;261:E737-741.
170. Tonkonogi M, Walsh B, Tiivel T, et al. Mitochondrial function in human skeletal muscle
is not impaired by high intensity exercise. Pflugers Arch. 1999;437:562-568.
171. Tesch PA, Thorsson A, Fujitsuka N. Creatine phosphate in fiber types of skeletal
muscle before and after exhaustive exercise. J Appl Physiol. 1989;66:1756-1759.
172. Colliander EB, Dudley GA, Tesch PA. Skeletal muscle fiber type composition and
performance during repeated bouts of maximal, concentric contractions. Eur J Appl Physiol
Occup Physiol. 1988;58:81-86.
173. Tesch PA. Muscle fatigue in man. With special reference to lactate accumulation
during short term intense exercise. Acta Physiologica Scandinavica. Supplementum,
1980;480:1-40.
174. Sun QA, Hess DT, Nogueira L, et al. Oxygen-coupled redox regulation of the skeletal
muscle ryanodine receptor-Ca2+ release channel by NADPH oxidase 4. Proc Natl Acad Sci U S
A. 2011;108:16098-16103.
175. Loureiro AC, do Rêgo-Monteiro IC, Louzada RA, et al. Differential expression of NADPH
oxidases depends on skeletal muscle fiber type in rats. Oxid Med Cell Longev.
2016;2016:6738701.
176. Edwards RH, Toescu V, Gibson H. Historical perspective: a framework for interpreting
pathobiological ideas on human muscle fatigue. In: Fatigue. Edited by Gandevia SC, Enoka
RM, McComas AJ et al., Springer, Boston, MA, 1995. pp. 481-494.
177. Saugen E, Vøllestad NK, Gibson H, et al. Dissociation between metabolic and
contractile responses during intermittent isometric exercise in man. Exp Physiol.
1997;82:213-26.
178. Wernbom M, Paulsen G, Nilsen TS, et al. Contractile function and sarcolemmal
permeability after acute low-load resistance exercise with blood flow restriction. Eur J Appl
Physiol. 2012; 112: 2051-2063.
179. Sieljacks P, Matzon A, Wernbom M, et al. Muscle damage and repeated bout effect
following blood flow restricted exercise. Eur J Appl Physiol. 2016;116:513-525.
180. Yasuda T, Brechue WF, Fujita T, et al. Muscle activation during low-intensity muscle
contractions with varying levels of external limb compression. J Sports Sci Med. 2008;7:467-
474.
181. Yasuda T, Brechue WF, Fujita T, et al. Muscle activation during low-intensity muscle
contractions with restricted blood flow. J Sports Sci. 2009;27:479-489.
182. Moritani T, Muro M, Kijima A, et al. Electromechanical changes during electrically
induced and maximal voluntary contractions: surface and intramuscular EMG responses
during sustained maximal voluntary contraction. Exp Neurol. 1985;88:484-499.
183. Sadamoto T, Bonde-Petersen F, Suzuki Y. Skeletal muscle tension, flow, pressure, and
EMG during sustained isometric contractions in humans. Eur J Appl Physiol Occup Physiol.
1983;51:395-408.
184. Sjøgaard G, Savard G, Juel C. Muscle blood flow during isometric activity and its
relation to muscle fatigue. Eur J Appl Physiol Occup Physiol. 1988;57:327-335.
185. Cupido CM, Galea V, McComas AJ. Potentiation and depression of the M wave in
human biceps brachii. J Physiol. 1996;491:541-550.

This article is protected by copyright. All rights reserved.


186. Galea V. Electrical characteristics of human ankle dorsi- and plantar-flexor muscles.
Comparative responses during fatiguing stimulation and recovery. Eur J Appl Physiol.
2001;85:130-140.
187. Leonard CT, Kane J, Perdaems J, et al. Neural modulation of muscle contractile
Accepted Article
properties during fatigue: afferent feedback dependence. Electroencephalogr Clin
Neurophysiol. 1994;93:209-217.
188. Hicks A, McComas AJ. Increased sodium pump activity following repetitive stimulation
of rat soleus muscles. J Physiol. 1989;414:337-349.
189. Rodriguez-Falces J, Place N. Determinants, analysis and interpretation of the muscle
compound action potential (M wave) in humans: implications for the study of muscle
fatigue. Eur J Appl Physiol. 2018;118:501-521.
190. Bigland-Ritchie B. EMG and fatigue of human voluntary and stimulated contractions.
In: Human Muscle Fatigue: Physiological Mechanisms. Chichester, UK: John Wiley & Sons,
Ltd., 1981. pp 130-156.
191. Dahlbäck LO, Ekstedt J, Stålberg E. Ischemic effects on impulse transmission to muscle
fibers in man. Electroencephalogr Clin Neurophysiol. 1970;29:579-591.
192. Sieck GC, Prakash YS. Fatigue at the neuromuscular junction. In: Fatigue. Edited by
Gandevia SC, Enoka RM, McComas AJ et al., Springer, Boston, MA, 1995. pp. 83-100.
193. Sandercock TG, Faulkner JA, Albers JW, Abbrecht PH. Single motor unit and fiber
action potentials during fatigue. J Appl Physiol. 1985;58:1073-1079.
194. Abramson DI, Rickert BL, Alexis JT, et al. Effect of repeated periods of ischemia on
motor nerve conduction velocity in forearm. J Appl Physiol. 1971;30:636-642.
195. Mittal P, Shenoy S, Sandhu JS. Effect of different cuff widths on the motor nerve
conduction of the median nerve: an experimental study. J Orthop Surg Res. 2008;3:1.
196. Desaulniers P, Lavoie PA, Gardiner PF. Incomplete recovery of endplate potential
amplitude while intermittently activating rat soleus neuromuscular junctions in situ. Muscle
Nerve. 2002;26:810-816.
197. Clausen T, Overgaard K, Nielsen OB. Evidence that the Na+-K+ leak/pump ratio
contributes to the difference in endurance between fast- and slow-twitch muscles. Acta
Physiol Scand. 2004;180:209-216.
198. Jansson E, Dudley GA, Norman B, Tesch PA. Relationship of recovery from intensive
exercise to the oxidative potential of skeletal muscle. Acta Physiol Scand. 1990;139:147-152.
199. Sjøgaard G, Houston ME, Nygaard E, Saltin B. Subgrouping of fast twitch fibres in
skeletal muscles of man. A critical appraisal. Histochemistry. 1978;58:79-87.
200. Staron RS, Hikida RS, Hagerman FC. Reevaluartion of human muscle fast-twitch
subtypes: evidence for a continuum. Histochemistry. 1983;78:33-39.
201. Staron RS, Hikida RS, Hagerman FC, et al. Human skeletal muscle fiber type
adaptability to various workloads. J Histochem Cytochem. 1984;32:146-152.
202. Essén-Gustavsson B, Henriksson J. Enzyme levels in pools of microdissected human
muscle fibres of identified type. Adaptive response to exercise. Acta Physiol Scand.
1984;120:505-515.
203. Sahlin K, Ren JM. Relationship of contraction capacity to metabolic changes during
recovery from a fatiguing contraction. J Appl Physiol. 1989;67:648-654.
204. Harris RC, Edwards RH, Hultman E, et al. The time course of phosphorylcreatine
resynthesis during recovery of the quadriceps muscle in man. Pflügers Arch. 1976;367:137-
142.
205. Umbel JD, Hoffman RL, Dearth DJ, et al. Delayed-onset muscle soreness induced by
low-load blood flow-restricted exercise. Eur J Appl Physiol. 2009 Dec; 107(6): 687-95.
206. Edström L, Kugelberg E. Histochemical composition, distribution of fibres and
fatiguability of single motor units. Anterior tibial muscle of the rat. J Neurol Neurosurg
Psychiatry. 1968;424-433.

This article is protected by copyright. All rights reserved.


207. Gollnick PD, Armstrong RB, Sembrowich WL, et al. Glycogen depletion pattern in
human skeletal muscle fibers after heavy exercise. J Appl Physiol. 1973;34:615-618.
208. Gollnick PD, Armstrong RB, Saubert CW 4th, et al. Glycogen depletion patterns in
human skeletal muscle fibers during prolonged work. Pflugers Arch. 1973;344:1-12.
Accepted Article
209. Gollnick PD, Piehl K, Saltin B. Selective glycogen depletion pattern in human muscle
fibres after exercise of varying intensity and at varying pedalling rates. J Physiol. 1974: 241:
45-57.
210. Vøllestad NK, Vaage O, Hermansen L. Muscle glycogen depletion patterns in type I and
subgroups of type II fibres during prolonged severe exercise in man.
Acta Physiol Scand, 1984: 122: 433-441.
211. Vøllestad NK, Blom PC. Effect of varying exercise intensity on glycogen depletion in
human muscle fibres. Acta Physiol Scand, 1985: 125: 395-405.
212. Vøllestad NK, Tabata I, Medbø JI. Glycogen breakdown in different human muscle
fibre types during exhaustive exercise of short duration. Acta Physiol Scand. 1992;144:135-
141.
213. Andersen JL, Gruschy-Knudsen T. Rapid switch-off of the human myosin heavy chain
IIX gene after heavy load muscle contractions is sustained for at least four days. Scand J Med
Sci Sports. 2018;28:371-380.
214. Robergs RA, Pearson DR, Costill DL, et al. Muscle glycogenolysis during differing
intensities of weight-resistance exercise. J Appl Physiol. 1991;70:1700-1706.
215. Pascoe DD, Gladden LB. Muscle glycogen resynthesis after short term, high intensity
exercise and resistance exercise. Sports Med. 1996;21:98-118.
216. Tesch PA, Ploutz-Snyder L., Yström L, et al. Skeletal muscle glycogen loss evoked by
resistance exercise. J Strength Cond Res. 1998:12:67-73.
217. Schmitz JP, Groenendaal W, Wessels B, et al. Combined in vivo and in silico
investigations of activation of glycolysis in contracting skeletal muscle. Am J Physiol Cell
Physiol. 2013;304:C180-193.
218. Jin ES, Sherry AD, Malloy CR. Evidence for reverse flux through pyruvate kinase in
skeletal muscle. Am J Physiol Endocrinol Metab. 2009;296:E748-757.
219. Jin ES, Sherry AD, Malloy CR. Lactate Contributes to Glyceroneogenesis and
Glyconeogenesis in Skeletal Muscle by Reversal of Pyruvate Kinase. J Biol Chem.
2015;290:30486-30497.
220. Lund J, Aas V, Tingstad RH, et al. Utilization of lactic acid in human myotubes and
interplay with glucose and fatty acid metabolism. Sci Rep. 2018;8:9814.
221. McLane JA, Holloszy JO. Glycogen synthesis from lactate in the three types of skeletal
muscle. J Biol Chem. 1979;254:6548-6553.
222. Beltman JG, Sargeant AJ, Haan H, et al. Changes in PCr/Cr ratio in single characterized
muscle fibre fragments after only a few maximal voluntary contractions in humans. Acta
Physiol Scand. 2004;180:187-193.
223. Beltman JG, de Haan A, Haan H, et al. Metabolically assessed muscle fibre recruitment
in brief isometric contractions at different intensities. Eur J Appl Physiol. 2004;92:485-492.
224. Tupling AR, Bombardier E, Stewart RD, et al. Muscle fiber type-specific response of
Hsp70 expression in human quadriceps following acute isometric exercise. J Appl Physiol.
2007;103:2105-2111.
225. Ingemann-Hansen T, Halkjaer-Kristensen J, Halskov O. Skeletal muscle phosphagen
and lactate concentrations in ischaemic dynamic exercise. Eur J Appl Physiol. 1981; 46: 261–
270.
226. Tesch P, Karlsson J. Lactate in fast and slow twitch skeletal muscle fibres of man
during isometric contraction. Acta Physiol Scand. 1977;99:230-236.
227. Johnson MA, Polgar J, Weightman D, Appleton D. Data on the distribution of fibre
types in thirty-six human muscles. An autopsy study. J Neurol Sci. 1973;18:111-129.

This article is protected by copyright. All rights reserved.


228. Elder GC, Bradbury K, Roberts R. Variability of fiber type distributions within human
muscles. J Appl Physiol. 1982;53:1473-1480.
229. Lexell J, Henriksson-Larsén K, Sjöström M. Distribution of different fibre types in
human skeletal muscles. 2. A study of cross-sections of whole m. vastus lateralis. Acta
Accepted Article
Physiol Scand. 1983;117:115-122.
230. Lexell J, Taylor CC. Variability in muscle fibre areas in whole human quadriceps
muscle. How much and why? Acta Physiol Scand. 1989;136:561-568.
231. Hultman E, Bergström J, Anderson NM. Breakdown and resynthesis of
phosphorylcreatine and adenosine triphosphate in connection with muscular work in man.
Scand J Clin Lab Invest. 1967;19:56-66.
232. Karlsson J, Saltin B. Lactate, ATP, and CP in working muscles during exhaustive
exercise in man. J Appl Physiol. 1970;29:596-602.
233. Karlsson J, Diamant B, Saltin B. Muscle metabolites during submaximal and maximal
exercise in man. Scand J Clin Lab Invest. 1970;26:385-394.
234. Rehunen S, Näveri H, Kuoppasalmi K, Härkönen M. High-energy phosphate
compounds during exercise in human slow-twitch and fast-twitch muscle fibres. Scand J Clin
Lab Invest. 1982;42:499-506.
235. Madapallimattam AG, Cross A, Nishio ML, Jeejeebhoy KN. Stability of high-energy
substrates in fast- and slow-twitch muscle: comparison of enzymatic assay of biopsy with in
vivo 31P nuclear magnetic resonance spectroscopy. Anal Biochem. 1994;217:103-139.
236. Farina D, Holobar A, Merletti R, Enoka RM. Decoding the neural drive to muscles from
the surface electromyogram. Clin Neurophysiol. 2010;121:1616-623.
237. Vigotsky AD, Halperin I, Lehman GJ, et al. Interpreting signal amplitudes in surface
electromyography studies in sport and rehabilitation sciences. Front Physiol. 2018;8:985.
238. Moritani T, Stegeman D, Merletti R. Basic physiology and biophysics of EMG signal
generation. In: Electromyography: physiology, engineering and noninvasive applications.
Edited by Merletti, R. and Parker, P. USA. IEEE Press. Wiley-lnterscience, 2004, pp. 1-25.
239. De Luca CJ, Chang SS, Roy SH, et al. Decomposition of surface EMG signals from cyclic
dynamic contractions. J Neurophysiol. 2015;113:1941-1951.
240. Farina D, Enoka RM. Surface EMG decomposition requires an appropriate validation. J
Neurophysiol. 2011;105:981-982.
241. De Luca CJ, Nawab H. Reply to Farina and Enoka: The reconstruct-and-test approach is
the most appropriate validation for surface EMG signal decomposition to date. J
Neurophysiol. 2011;105:983-984.
242. Farina D, Merletti R, Enoka RM. The extraction of neural strategies from the surface
EMG: an update. J Appl Physiol. 2014;117:1215-1230.
243. Farina D, Merletti R, Enoka RM. Reply to De Luca, Nawab, and Kline: The proposed
method to validate surface EMG signal decomposition remains problematic. J Appl Physiol.
2015;118:1085.
244. De Luca CJ, Nawab SH, Kline JC. Clarification of methods used to validate surface EMG
decomposition algorithms as described by Farina et al. (2014). J Appl Physiol. 2015;118:1084.
245. Arabadzhiev TI, Dimitrov VG, Dimitrova NA, Dimitrov GV. Influence of motor unit
synchronization on amplitude characteristics of surface and intramuscularly recorded EMG
signals. Eur J Appl Physiol. 2010;108:227-237.
246. Arabadzhiev TI, Dimitrov VG, Dimitrov GV. The increase in surface EMG could be a
misleading measure of neural adaptation during the early gains in strength. Eur J Appl
Physiol. 2014;114:1645-1655.
247. Dimitrova NA, Dimitrov GV. Interpretation of EMG changes with fatigue: facts, pitfalls,
and fallacies. J Electromyogr Kinesiol. 2003;13:13-36.
248. Hanson J, Persson A. Changes in the action potential and contraction of isolated frog
muscle after repetitive stimulation. Acta Physiol Scand. 1971;81:340-348.

This article is protected by copyright. All rights reserved.


249. Hanson J. The effects of repetitive stimulation on the action potential and the twitch
of rat muscle. Acta Physiol Scand. 1974;90:387-400.
250. Hanson J. Effects of repetitive stimulation on membrane potentials and twitch in
human and rat intercostal muscle fibres. Acta Physiol Scand. 1974;92:238-248.
Accepted Article
251. Keenan KG, Farina D, Maluf KS, et al. Influence of amplitude cancellation on the
simulated surface electromyogram. J Appl Physiol. 2005;98:120-131.
252. Dideriksen JL, Enoka RM, Farina D. Neuromuscular adjustments that constrain
submaximal EMG amplitude at task failure of sustained isometric contractions. J Appl
Physiol. 2011;111:485-494.
253. Farina D, Merletti R, Enoka RM. The extraction of neural strategies from the surface
EMG. J Appl Physiol. 2004;96:1486-1495.
254. Neyroud D, Kayser B, Place N. The need to normalize surface EMG. J Appl Physiol.
2015;119:1519.
255. Martinez-Valdes E, Negro F, Falla D, et al. Surface electromyographic amplitude does
not identify differences in neural drive to synergistic muscles. J Appl Physiol. 2018;124:1071-
1079.
256. Moritani T, Muro M. Motor unit activity and surface electromyogram power spectrum
during increasing force of contraction. Eur J Appl Physiol Occup Physiol. 1987;56:260-265.
257. Clamann HP. Activity of single motor units during isometric tension. Neurology.
1970;20:254-260.
258. Knight CA, Kamen G. Superficial motor units are larger than deeper motor units in
human vastus lateralis muscle. Muscle Nerve. 2005;31:475-480.
259. Edgerton VR, Smith JL, Simpson DR. Muscle fibre type populations of human leg
muscles. Histochem J. 1975;7:259-266.
260. Ray CA, Dudley GA. Muscle use during dynamic knee extension: implication for
perfusion and metabolism. J Appl Physiol. 1998;85:1194-1197.
261. Enocson AG, Berg HE, Vargas R, et al. Signal intensity of MR-images of thigh muscles
following acute open- and closed chain kinetic knee extensor exercise - index of muscle use.
Eur J Appl Physiol. 2005;94:357-363.
262. Kalliokoski KK, Boushel R, Langberg H, et al. Differential glucose uptake in quadriceps
and other leg muscles during one-legged dynamic submaximal knee-extension exercise.
Front Physiol. 2011;2:75.
263. Richardson RS, Frank LR, Haseler LJ. Dynamic knee-extensor and cycle exercise:
functional MRI of muscular activity. Int J Sports Med. 1998;19:182-187.
264. Watanabe K, Akima H. Normalized EMG to normalized torque relationship of vastus
intermedius muscle during isometric knee extension. Eur J Appl Physiol. 2009;106:665-673.
265. Thorstensson A, Karlsson J, Viitasalo JH, et al. Effect of strength training on EMG of
human skeletal muscle. Acta Physiol Scand. 1976;98:232-236.
266. Häkkinen K, Komi PV. Electromyographic changes during strength training and
detraining. Med. Sci. Sports Exerc. 1983;15:455-460.
267. Eloranta V. Patterning of muscle activity in static knee extension. Electromyogr. Clin.
Neurophysiol. 1989;29:369-375.
268. Alkner BA, Tesch PA, Berg HE. Quadriceps EMG/force relationship in knee extension
and leg press. Med Sci Sports Exerc. 2000;32:459-463.
269. Rabita G, Pérot C, Lensel-Corbeil G. Differential effect of knee extension isometric
training on the different muscles of the quadriceps femoris in humans. Eur J Appl Physiol.
2000;83:531-538.
270. Amiridis IG, Martin A, Morlon B, et al. Co-activation and tension-regulating
phenomena during isokinetic knee extension in sedentary and highly skilled humans. Eur J
Appl Physiol Occup Physiol. 1996;73:149-156.

This article is protected by copyright. All rights reserved.


271. Komi PV, Linnamo V, Silventoinen P, Sillanpää M. Force and EMG power spectrum
during eccentric and concentric actions. Med Sci Sports Exerc. 2000;32:1757-1762.
272. Ekstrom RA, Osborn RW, Goehner HM, et al. Electromyographic normalization
procedures for determining exercise intensity of closed chain exercises for strengthening the
Accepted Article
quadriceps femoris muscles. J Strength Cond Res. 2012;26:766-771.
273. Beutler AI, Cooper LW, Kirkendall DT, Garrett WE Jr. Electromyographic analysis of
single-leg, closed chain exercises: implications for rehabilitation after anterior cruciate
ligament reconstruction. J Athl Train. 2002;37:13-18.
274. Westing SH, Cresswell AG, Thorstensson A. Muscle activation during maximal
voluntary eccentric and concentric knee extension. Eur J Appl Physiol Occup Physiol.
1991;62:104-108.
275. Aagaard P, Simonsen EB, Andersen JL, et al. Neural inhibition during maximal
eccentric and concentric quadriceps contraction: effects of resistance training. J Appl Physiol.
2000;89:2249-2257.
276. Tesch PA, Dudley GA, Duvoisin MR, et al. Force and EMG signal patterns during
repeated bouts of concentric or eccentric muscle actions. Acta Physiol Scand. 1990;138:263-
721.
277. Duchateau J, Enoka RM. Human motor unit recordings: origins and insight into the
integrated motor system. Brain Res. 2011;1409:42-61.
278. Basmajian JV, De Luca CJ. Muscles Alive. Baltimore: Williams & Wilkins, 1985.
279. Moritani T, Muro M, Nagata A. Intramuscular and surface electromyogram changes
during muscle fatigue. J Appl Physiol. 1986;60:1179-1185.
280. Trontelj JV, Jabre J, Mihelin M. Needle and wire detection techniques. In:
Electromyography: physiology, engineering and noninvasive applications. Edited by Merletti,
R. and Parker, P. USA. IEEE Press. Wiley-lnterscience, 2004, 27-46.
281. Kent-Braun JA, Miller RG, Weiner MW. Human skeletal muscle metabolism in health
and disease: utility of magnetic resonance spectroscopy. Exerc Sport Sci Rev. 1995;23:305-
347.
282. Sullivan MJ, Saltin B, Negro-Vilar R, et al. Skeletal muscle pH assessed by biochemical
and 31P-MRS methods during exercise and recovery in men. J Appl Physiol. 1994;77:2194-
3200.
283. Constantin-Teodosiu D, Greenhaff PL, et al. Anaerobic energy production in human
skeletal muscle in intense contraction: a comparison of 31P magnetic resonance
spectroscopy and biochemical techniques. Exp Physiol. 1997;82:593-601.
284. Vandenborne K, McCully K, Kakihira H, et al. Metabolic heterogeneity in human calf
muscle during maximal exercise. Proc Natl Acad Sci U S A. 1991;88:5714-5718.
285. Park JH, Brown RL, Park CR, et al. Functional pools of oxidative and glycolytic fibers in
human muscle observed by 31P magnetic resonance spectroscopy during exercise. Proc Natl
Acad Sci U S A. 1987;84:8976-8980.
286. Achten E, Van Cauteren M, Willem R, et al. 31P-NMR spectroscopy and the metabolic
properties of different muscle fibers. J Appl Physiol. 1990;68:644-659.
287. Houtman CJ, Heerschap A, Zwarts MJ, Stegeman DF. pH heterogeneity in tibial
anterior muscle during isometric activity studied by (31)P-NMR spectroscopy. J Appl Physiol.
2001;91:191-200.
288. Harris RC, Essén B, Hultman E. Glycogen phosphorylase activity in biopsy samples and
single muscle fibres of musculus quadriceps femoris of man at rest. Scand J Clin Lab Invest.
1976;36:512-516.
289. Mizuno M, Secher NH, Quistorff B. 31P-NMR spectroscopy, rsEMG, and histochemical
fiber types of human wrist flexor muscles. J Appl Physiol. 1994;76:531-538.

This article is protected by copyright. All rights reserved.


290. Sahlin K, Harris RC, Hultman E. Creatine kinase equilibrium and lactate content
compared with muscle pH in tissue samples obtained after isometric exercise. Biochem J.
1975;152:173-180.
291. Meyer RA, Slade JM, Towse TF, et al. Phosphocreatine resynthesis during recovery
Accepted Article
after exercise with blood flow occlusion. Med Sci Sports Exerc 2008;40.5: S349.
292. Taylor DJ, Bore PJ, Styles P, et al. Bioenergetics of intact human muscle. A 31P nuclear
magnetic resonance study. Mol Biol Med. 1983;1:77-94.
293. Jeneson JA, Wesselink MW, de Boer RW, Amelink HG. Peak-splitting of inorganic
phosphate during exercise: anatomy or physiology? A MRI-guided 31P MRS study of human
forearm muscle. In: Proc. 8th A. Meet. Soc. Magn. Reson. Med. 1989. p. 1030.
294. Johnson R, Arnold D, Leger G. Splitting of the phosphate peak in exercising muscle is
due to macroscopic not microscopic heterogeneity (Abstract). Magn. Reson. Med.
1990;Suppl. 2: 866.
295. Vandenborne K, Walter G, Leigh JS, Goelman G. pH heterogeneity during exercise in
localized spectra from single human muscles. Am J Physiol. 1993;265:C1332-1339.
296. Mizuno M, Justesen LO, Bedolla J, et al. Partial curarization abolishes splitting of the
inorganic phosphate peak in 31P-NMR spectroscopy during intense forearm exercise in man.
Acta Physiol Scand. 1990;139:611-2.
297. Mizuno M, Horn A, Secher NH, Quistorff B. Exercise-induced 31P-NMR metabolic
response of human wrist flexor muscles during partial neuromuscular blockade. Am J
Physiol. 1994;267:R408-414.
298. Yanagisawa O, Niitsu M, Yoshioka H, et al. MRI determination of muscle recruitment
variations in dynamic ankle plantar flexion exercise. Am J Phys Med Rehabil. 2003;10:760-
765.
299. Segal RL, Song AW. Nonuniform activity of human calf muscles during an exercise
task. Arch Phys Med Rehabil. 2005;86:2013-2017.
300. Meyerspeer M, Robinson S, Nabuurs CI, et al. Comparing localized and nonlocalized
dynamic 31P magnetic resonance spectroscopy in exercising muscle at 7 T. Magn Reson
Med. 2012;68:1713-1723.
301. Valkovič L, Chmelík M, Just Kukurová I, et al. Depth-resolved surface coil MRS (DRESS)-
localized dynamic (31) P-MRS of the exercising human gastrocnemius muscle at 7 T. NMR
Biomed. 2014;27:1346-1352.
302. Rossiter HB, Ward SA, Howe FA, et al. Dynamics of intramuscular 31P-MRS P(i) peak
splitting and the slow components of PCr and O2 uptake during exercise. J Appl Physiol.
2002;93:2059-2069.
303. Tesch PA, Karlsson J. Effects of exhaustive, isometric training on lactate accumulation
in different muscle fiber types. Int J Sports Med. 1984;5:89-91.
304. Houtman CJ, Heerschap A, Zwarts MJ, Stegeman DF. An additional phase in PCr use
during sustained isometric exercise at 30% MVC in the tibialis anterior muscle. NMR Biomed.
2002;15:270-277.
305. Yoshida T, Watari H. Exercise-induced splitting of the inorganic phosphate peak:
investigation by time-resolved 31P-nuclear magnetic resonance spectroscopy. Eur J Appl
Physiol Occup Physiol. 1994;69:465-473.
306. Miller RG, Kent-Braun JA, Sharma KR, Weiner MW. Mechanisms of human muscle
fatigue. Quantitating the contribution of metabolic factors and activation impairment. In:
Fatigue. Edited by Gandevia SC, Enoka RM, McComas AJ et al., Springer, Boston, MA, 1995.
pp. 195-201.
307. Miller RG, Giannini D, Milner-Brown HS, et al. Effects of fatiguing exercise on high-
energy phosphates, force, and EMG: evidence for three phases of recovery. Muscle Nerve.
1987;10:810-821.

This article is protected by copyright. All rights reserved.


308. Vestergaard-Poulsen P, Thomsen C, Sinkjaer T, et al. Simultaneous electromyography
and 31P nuclear magnetic resonance spectroscopy--with application to muscle fatigue.
Electroencephalogr Clin Neurophysiol. 1992;85:402-411.
309. Vestergaard-Poulsen P, Thomsen C, et al. Simultaneous 31P-NMR spectroscopy and
Accepted Article
EMG in exercising and recovering human skeletal muscle: a correlation study. J Appl Physiol.
1995;79:1469-1478.
310. Bendahan D, Jammes Y, Salvan AM. Combined electromyography--31P-magnetic
resonance spectroscopy study of human muscle fatigue during static contraction. Muscle
Nerve. 1996;19:715-721.
311. Houtman CJ, Stegeman DF, Van Dijk JP, Zwarts MJ. Changes in muscle fiber
conduction velocity indicate recruitment of distinct motor unit populations. J Appl Physiol.
2003;95:1045-1054.
312. Giannesini B, Cozzone PJ, Bendahan D. Non-invasive investigations of muscular
fatigue: metabolic and electromyographic components. Biochimie. 2003;85:873-883.
313. Crenshaw AG, Hargens AR, Gershuni DH, Rydevik B. Wide tourniquet cuffs more
effective at lower inflation pressures. Acta Orthop Scand 1988: 59: 447-451.
314. Wernbom M, Augustsson J, Thomeé R. Effects of vascular occlusion on muscular
endurance in dynamic knee extension exercise at different submaximal loads. J Strength
Cond Res. 2006;20:372-377.
315. Brooke MH, Kaiser KK. Muscle fiber types: how many and what kind? Arch Neurol.
1970;23:369-739.
316. Green H, Goreham C, Ouyang J, et al. Regulation of fiber size, oxidative potential, and
capillarization in human muscle by resistance exercise. Am J Physiol. 1999;276:R591-596.
317. Billeter R, Heizmann CW, Howald H, Jenny E. Analysis of myosin light and heavy chain
types in single human skeletal muscle fibers. Eur J Biochem. 1981;116:389-395.
318. Brooke MH, Kaiser KK. Three "myosin adenosine triphosphatase" systems: the nature
of their pH lability and sulfhydryl dependence. J Histochem Cytochem. 1970;18:670-672.
319. Dubowitz V, Sewry CA, Oldfors A. Muscle biopsy: a practical approach. 4th Edition.
Elsevier Health Sciences, 2013.
320. Fridén J, Seger J, Ekblom B. Sublethal muscle fibre injuries after high-tension
anaerobic exercise. Eur J Appl Physiol Occup Physiol. 1988;57:360-368.
321. Han YS, Proctor DN, Geiger PC, Sieck GC. Reserve capacity for ATP consumption during
isometric contraction in human skeletal muscle fibers. J Appl Physiol. 2001;90:657-664.
322. Piehl K. Time course for refilling of glycogen stores in human muscle fibres following
exercise-induced glycogen depletion. Acta Physiol Scand. 1974;90:297-302.
323. Fairchild TJ, Armstrong AA, Rao A. Glycogen synthesis in muscle fibers during active
recovery from intense exercise. Med Sci Sports Exerc. 2003;35:595-602.
324. Wernbom M, Apro W, Paulsen G, et al. Acute low-load resistance exercise with and
without blood flow restriction increased protein signalling and number of satellite cells in
human skeletal muscle. Eur J Appl Physiol. 2013;113:2953-2965.
325. Zehnder M, Muelli M, Buchli R, et al. Further glycogen decrease during early recovery
after eccentric exercise despite a high carbohydrate intake. Eur J Nutr. 2004;43:148-159.
326. Andersen P, Saltin B. Maximal perfusion of skeletal muscle in man. J Physiol.
1985;366:233-249.
327. Person RS, Golubovich K. Electromyographic study of fatigue in man in conditions of
artificial ischemia in the working muscle. Fiziologicheskii zhurnal SSSR imeni IM Sechenova,
1960;46:1181-1187.
328. Arendt-Nielsen L, Mills KR. The relationship between mean power frequency of the
EMG spectrum and muscle fibre conduction velocity. Electroencephalogr Clin Neurophysiol.
1985;60:130-134.

This article is protected by copyright. All rights reserved.


329. Myers SJ, Sullivan WP. Effect of crculatory occlusion on time to muscular fatigue. J
Appl Physiol. 1968;24:54-59.
330. Sullivan WP. Muscle fatigue and electromyographic changes during isometric exercise
in presence of vascular occlusion. 1968. PhD Thesis. The Ohio State University.
Accepted Article
331. Edgerton VR, Roy RR, Apor P. Specific tension of human elbow flexor muscles. In:
Saltin B (ed) Biochemistry of exercise VI. Human Kinetics, Champaign, Ill., 1986, pp 487-500.
332. Signorile JF, Digel S, Moccia G, et al. Effects of partial occlusion of circulation on
frequency and amplitude of surface electromyography. J Electromyogr Kinesiol. 1991;1:124-
129.
333. Yasuda T, Fukumura K, Iida H, Nakajima T. Effect of low-load resistance exercise with
and without blood flow restriction to volitional fatigue on muscle swelling. Eur J Appl Physiol.
2015;115:919-926.
334. Alway SE, MacDougall JD, Sale DG. Contractile adaptations in the human triceps surae
after isometric exercise. J Appl Physiol. 1989;66:2725-2732.
335. Alway SE, Sale DG, MacDougall JD. Twitch contractile adaptations are not dependent
on the intensity of isometric exercise in the human triceps surae. Eur J Appl Physiol Occup
Physiol. 1990;60:346-352.
336. Moore DR, Burgomaster KA, Schofield LM, et al. Neuromuscular adaptations in human
muscle following low intensity resistance training with vascular occlusion. Eur J Appl Physiol.
2004;92:399-406.
337. Borg G. Borg's perceived exertion and pain scales. Human Kinetics, Champaign, Ill.,
1998.
338. Kooistra RD, de Ruiter CJ, de Haan A. Muscle activation and blood flow do not explain
the muscle length-dependent variation in quadriceps isometric endurance. J Appl Physiol.
2005;98:810-816.
339. Cook SB, Murphy BG, Labarbera KE. Neuromuscular function after a bout of low-load
blood flow-restricted exercise. Med Sci Sports Exerc. 2013;45:67-74.
340. Wernbom M. (2011). Effects of an acute bout of low-load resistance training with
blood flow restriction - with special reference to muscle damage, hypertrophic signaling and
satellite cells (Doctoral dissertation, Norwegian School of Sport Sciences, Oslo, Norway).
Retrieved from Google Scholar.
341. Loenneke JP, Kim D, Fahs CA, et al. Effects of exercise with and without different
degrees of blood flow restriction on torque and muscle activation. Muscle Nerve 2015;713-
721.
342. Fatela P, Reis JF, Mendonca GV, et al. Acute neuromuscular adaptations in response
to low-intensity blood-flow restricted exercise and high-intensity resistance exercise: are
there any differences? J Strength Cond Res. 2018;32:902-910.
343. Magora A, Blank A, Gonen B. Effects of artificially induced ischemia (AII) on the
electrophysiological pattern of muscular fatigue in healthy humans. Electromyogr Clin
Neurophysiol. 1980;20:125-140.
344. Yoshida T, Watari H. Effect of circulatory occlusion on human muscle metabolism
during exercise and recovery. Eur J Appl Physiol Occup Physiol. 1997;75:200-205.
345. Suga T, Okita K, Morita N, et al. Intramuscular metabolism during low-intensity
resistance exercise with blood flow restriction. J Appl Physiol. 2009;106:1119-1124.
346. Suga T, Okita K, Morita N, et al. Dose effect on intramuscular metabolic stress during
low-intensity resistance exercise with blood flow restriction. J Appl Physiol . 2010;108:1563-
1567.
347. Trappe TA, Raue U, Tesch PA. Human soleus muscle protein synthesis following
resistance exercise. Acta Physiol Scand. 2004;182:189-196.
348. Gravel D, Richards CL, Filion M. Angle dependency in strength measurements of the
ankle plantar flexors. Eur J Appl Physiol Occup Physiol. 1990;61:182-187.

This article is protected by copyright. All rights reserved.


349. Peeters M, Svantesson U, Grimby G. Effect of prior isometric muscle action on
concentric torque output during plantar flexion. Eur J Appl Physiol Occup Physiol.
1995;71:272-275.
350. Nadeau S, Gravel D, Arsenault AB, Goyette M. Preloading and range of motion effect
Accepted Article
on plantarflexor muscle performance. Arch Phys Med Rehabil. 1996;77:1000-1004.
351. Svantesson U, Grimby G, Thomeé R. Potentiation of concentric plantar flexion torque
following eccentric and isometric muscle actions. Acta Physiol Scand. 1994;152:287-293.
352. Yanagisawa O, Sanomura M. Effects of low-load resistance exercise with blood flow
restriction on high-energy phosphate metabolism and oxygenation level in skeletal muscle.
Interv Med Appl Sci. 2017;9:67-75.
353. Saltin B, Gollnick PD. Skeletal muscle adaptability: significance for metabolism and
performance. Handbook of Physiology. Skeletal Muscle, 1983,10:555-631.
354. Bangsbo J, Johansen L, Quistorff B, Saltin B. NMR and analytic biochemical evaluation
of CrP and nucleotides in the human calf during muscle contraction. J Appl Physiol.
1993;74:2034-2039.
355. Vandenborne K, Walter G, Ploutz-Snyder L, et al. Energy-rich phosphates in slow and
fast human skeletal muscle. Am J Physiol. 1995;268:C869-876.
356. Takarada Y, Sato Y, Ishii N. Effects of resistance exercise combined with vascular
occlusion on muscle function in athletes. Eur J Appl Physiol 2002: 86: 308-314.
357. Kawada S. What phenomena do occur in blood flow-restricted muscle? Int J Kaatsu
Training Res 2005: 1: 37-44.
358. Grimby L, Hannerz J. Disturbances in voluntary recruitment order of low and high
frequency motor units on blockades of proprioceptive afferent activity. Acta Physiol Scand.
1976;96:207-216.
359. Buchthal F, Pinell P, Rosenfalck P. Action potential parameters in normal human
muscle and their physiological determinants. Acta Physiol Scand. 1954;32:219-229.
360. Goldberg J, Sullivan SJ, Seaborne DE. The effect of two intensities of massage on H-
reflex amplitude. Phys Ther. 1992;72:449-457.
361. Robichaud JA, Agostinucci J, Vander Linden DW. Effect of air-splint application on
soleus muscle motoneuron reflex excitability in nondisabled subjects and subjects with
cerebrovascular accidents. Phys Ther. 1992;72:176-183.
362. Agostinucci J. Circumferential pressure’s inhibitory effects on soleus H-reflex. Transl
Neurosci 2013;4:251-259.
363. Agostinucci J. The effects of circumferential pressure on the soleus muscle F-wave in
healthy subjects. J Electromyogr Kinesiol. 2012;22:223-227.
364. Ge W, Khalsa PS. Encoding of compressive stress during indentation by group III and
IV muscle mechano-nociceptors in rat gracilis muscle. J Neurophysiol. 2003;89:785-792.
365. Ørtenblad N, Macdonald WA, Sahlin K. Glycolysis in contracting rat skeletal muscle is
controlled by factors related to energy state. Biochem J. 2009;420:161-168.
366. Lowe DA, Alway SE. Animal models for inducing muscle hypertrophy: are they
relevant for clinical applications in humans? J Orthop Sports Phys Ther. 2002;32:36-43.
367. Goto K, Okuyama R, Sugiyama H, et al. Effects of heat stress and mechanical stretch
on protein expression in cultured skeletal muscle cells. Pflügers Arch. 2003:447:247-253.
368. Goto K, Oda H, Kondo H, et al. Responses of muscle mass, strength and gene
transcripts to long-term heat stress in healthy human subjects. Eur J Appl Physiol.
2011;111:17-27.
369. Sudo M, Ando S, Kano Y. Repeated blood flow restriction induces muscle fiber
hypertrophy. Muscle Nerve. 2017;55:274-276.
370. Natsume T, Ozaki H, Saito AI, et al. Effects of electrostimulation with blood flow
restriction on muscle size and strength. Med Sci Sports Exerc. 2015;47:2621-2627.

This article is protected by copyright. All rights reserved.


371. Gorgey AS, Timmons MK, Dolbow DR, et al. Electrical stimulation and blood flow
restriction increase wrist extensor cross-sectional area and flow meditated dilatation
following spinal cord injury. Eur J Appl Physiol. 2016;116:1231-1244.
372. Nakajima T, Koide S, Yasuda T, et al. Muscle hypertrophy following blood flow-
Accepted Article
restricted, low-force isometric electrical stimulation in rat tibialis anterior: role for muscle
hypoxia. J Appl Physiol. 2018;125:134-145.
373. Slysz JT, Burr JF. The effects of blood flow restricted electrostimulation on strength
and hypertrophy. J Sport Rehabil. 2018;27:257-262.
374. Goto K, Oda H, Morioka S, et al. Skeletal muscle hypertrophy induced by low-intensity
exercise with heat stress in healthy human subjects. Jpn J Aerosp Environ Med. 2007;44:13-
18.
375. Wernbom M, Augustsson J, Thomeé R. The influence of frequency, intensity, volume
and mode of strength training on whole muscle cross-sectional area in humans. Sports Med.
2007; 37: 225-264.
376. Mitchell CJ, Churchward-Venne TA, West DW, et al. Resistance exercise load does not
determine training-mediated hypertrophic gains in young men. J Appl Physiol. 2012;113:71-
77.
377. Sieljacks P, Degn R, Hollaender K, et al. Non-failure blood flow restricted exercise
induces similar muscle adaptations and less discomfort than failure protocols. Scand J Med
Sci Sports. 2019;29:336-347.
378. Kubo K, Komuro T, Ishiguro N, et al. Effects of low-load resistance training with
vascular occlusion on the mechanical properties of muscle and tendon. J Appl Biomech.
2006;22:112-119.
379. Colomer-Poveda D, Romero-Arenas S, Vera-Ibáñez A, et al. Effects of 4 weeks of low-
load unilateral resistance training, with and without blood flow restriction, on strength,
thickness, V wave, and H reflex of the soleus muscle in men. Eur J Appl Physiol.
2017;117:1339-1347.
380. Cook SB, Scott BR, Hayes KL, Murphy BG. Neuromuscular Adaptations to low-load
blood flow restricted resistance training. J Sports Sci Med. 2018;17:66-73.

This article is protected by copyright. All rights reserved.


Figure legends.
Accepted Article
Figure 1. Suggested scheme of the interplay between the recruitment of motor units
innervating type I, IIA, IIAX and IIX muscle fibres and the accompanying increases in firing
rates with increases in contractile force production. The scheme depicts short (~0.5-1 s) but
non-ballistic contractions, as opposed to slow ramp (~10 s) contractions (see text). Modified
from Sale42 based on data discussed in this review.

Figure 2. Some of the possible pathways and processes of fatigue in strenuous BFRRE. See
text for details. Thicker lines indicate a relatively more substantial influence.

Figure 3. Neighbouring muscle cross sections stained with the PAS staining method for
glycogen (left) and for MHC I (right, bright red staining) to display type I fibres, showing
lower glycogen levels in type I fibres at 1 hour after an acute bout of BFRRE in one subject.
Figure courtesy of Kristoffer Cumming, with permission.

Figure 4. Nonconsecutive muscle cross sections stained with the PAS staining method for
glycogen (left) and for MHC I (right, green staining) to display type I fibres, showing lower
glycogen levels in type I fibres at 24 hours after an acute bout of BFRRE in one subject.
Figure courtesy of Kristoffer Cumming and Mathias Wernbom.

Figures 5a-d. Muscle activity measured by EMG during an acute bout of BFRRE exercise
consisting of 4 sets to volitional failure of low-load dynamic knee extensions (20-25% of
1RM) with continuous partial BFR (~50% of seated resting arterial occlusion pressure). Rest
between the sets: 45 seconds. Upper left figure: 1st set, 61 repetitions. Lower right figure: 4th
set, 8 repetitions. EMG normalised to MVC (100%). Red = vastus lateralis, left leg. Blue =
vastus medialis, left leg. Figures adapted from Wernbom340.

Figure 6. Muscle EMG activity during heavy resistance exercise bout with dynamic knee
extensions (80-85% of 1RM), a single set to failure (7 repetitions). Same subject as in Figure
6. EMG normalised to isometric MVC (100%). Red = vastus lateralis, right leg. Blue = vastus
medialis, right leg. Adapted from Wernbom340.

Figure 7. Suggested scheme of the successive and cumulative activation of type I, IIA, IIAX
and IIX muscle fibres in low-load BFRRE with increasing fatigue and duration. The scheme
depicts BFRRE performed at ~20-30% of 1RM with a hypothetical representative limb
muscle (e.g. the vastus lateralis or the biceps brachii) consisting of 50% type I, 30% type IIA,
10% type IIAX and 10% type IIX muscle fibres. The scheme reflects the proposed largely
intact orderly recruitment of motor units in BFRRE, but also allows for slight alterations in
recruitment thresholds that may occur in some motor units (see text for details).

This article is protected by copyright. All rights reserved.


Accepted Article

This article is protected by copyright. All rights reserved.


Accepted Article

This article is protected by copyright. All rights reserved.


Accepted Article

This article is protected by copyright. All rights reserved.


Accepted Article

This article is protected by copyright. All rights reserved.


Accepted Article

This article is protected by copyright. All rights reserved.


Accepted Article

This article is protected by copyright. All rights reserved.


Accepted Article

This article is protected by copyright. All rights reserved.