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Blood flow restriction does not alter the early hypertrophic signaling and
short-term adaptive response to resistance exercise when performed to task
failure

Article in Journal of Applied Physiology: Respiratory, Environmental and Exercise Physiology · April 2023
DOI: 10.1152/japplphysiol.00529.2022

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 J Appl Physiol 134: 1265–1277, 2023.
First published April 13, 2023; doi:10.1152/japplphysiol.00529.2022

RESEARCH ARTICLE

Blood flow restriction does not alter the early hypertrophic signaling and short-
term adaptive response to resistance exercise when performed to task failure
Christopher Pignanelli, Graham P. Holloway, and Jamie F. Burr
Department of Human Health & Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada

Abstract
Previous research supports that low-load resistance exercise with blood flow restriction (LL-BFR) acutely increases physiological
responses and muscle mass accrual compared with low-load resistance exercise (LL-RE) alone. However, most studies have
work-matched LL-BFR and LL-RE. Completing sets to similar perceived efforts, thereby allowing for a variable amount of work,
may provide a more ecologically valid approach to compare LL-BFR and LL-RE. This study aimed to examine acute signaling and
training responses following LL-RE or LL-BFR performed to task failure. Ten participants had each leg randomly assigned to per-
form LL-RE or LL-BFR. Muscle biopsies were obtained before and 2-h after the first exercise bout and after 6-wk of training for
Western blot and immunohistochemistry analyses. Repeated measure ANOVA and intraclass coefficients (ICCs) were used to
compare responses of each condition. After exercise, AKT(T308) phosphorylation increased after LL-RE and LL-BFR (both
145%
of baseline, P < 0.05) and trended for p70 S6K(T389) (LL-RE:
158% and LL-BFR:
137%, P = 0.06). BFR did not alter these
responses, resulting in fair-excellent ICCs for signaling proteins involved in anabolism (ICCAKT(T308) = 0.889, P = 0.001;
ICCAKT(S473) = 0.519, P = 0.074; ICCp70 S6K(T389) = 0.514, P = 0.105). After training, muscle fiber cross-sectional area and
vastus lateralis whole muscle thickness were similar between conditions (ICC  0.637, P  0.031). Similar acute and chronic
responses between conditions and high ICC values between legs suggest that both LL-BFR and LL-RE performed by the
same person result in similar adaptations. These data support the concept that sufficient muscular exertion is a key factor
for training-induced muscle hypertrophy with low-load resistance exercise independent of total work and blood flow.
NEW & NOTEWORTHY The addition of blood flow restriction during low-load resistance exercise is considered to increase the
signaling events and muscle growth responses to a greater extent than low-load resistance exercise alone. It remains unclear
whether blood flow restriction accelerates or increases these adaptive responses, as most studies have each condition perform
the same amount of work. Despite different amounts of work performed, we show similar signaling and muscle growth
responses occur after low-load resistance exercise with and without blood flow restriction. Our work supports that blood flow
restriction accelerates fatigue but does not increase the signaling events and muscle growth responses during low-load resist-
ance exercise.

ischemia; maximal repetitions; oxygen availability; volitional fatigue

INTRODUCTION 30  15  15  15 repetitions) or matched to the number of

Low-load resistance exercise with blood flow restriction
(LL-BFR) has been shown to induce greater muscular hyper-
trophy than low-load resistance exercise (LL-RE) with con-
served blood flow (1–4). Research in the past two decades
has mechanistically supported the observation that LL-BFR
increases the acute anabolic responses that are typically
superior to LL-RE. For example, LL-BFR alters the phospho-
rylation status of signaling proteins implicated in gene tran-
scription and/or translation regulation (5–9) and increases
the rate of muscle protein synthesis to a greater extent than
LL-RE in the initial hours-days after a bout of exercise (5, 6,
8, 10). A caveat to these findings is that LL-RE is typically
“volume-matched” (i.e., similar load  repetitions) to LL-
BFR using arbitrary repetition schemes (e.g., four sets of
repetitions until task failure with LL-BFR. A consequence of
volume-matched designs is that BFR exercise elicits greater
perceived effort (11), compared with the free-flow condition.
This effort is likely to be accompanied by greater muscle
activation via fiber recruitment and/or firing rate, as sup-
ported by direct intramuscular electromyographic (EMG)
recordings (12) and changes in individual muscle fiber
metabolite concentrations (13). Greater muscle activation
through processes associated with excitation-contraction
coupling could in part explain the increased anabolic
responses observed with exercise under reduced blood flow.
This is because intracellular calcium can initiate cell signal-
ing events (14) and the muscle growth response may involve
calcium-dependent pathways (15). However, reducing mus-
cle oxygen availability per se can also activate calcium

Correspondence: J. F. Burr (burrj@uoguelph.ca).

Submitted 8 September 2022 / Revised 4 April 2023 / Accepted 4 April 2023

http://www.jap.org 8750-7587/23 Copyright © 2023 the American Physiological Society. 1265

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 RESISTANCE TRAINING TO TASK FAILURE WITH AND WITHOUT BFR
signaling pathways (16). Thus, volume-matched designs
make it challenging to fully understand if the observed cell
signaling and anabolic responses with BFR exercise are due
to increased muscle activation and/or are a direct conse-
quence of reduced muscle oxygen availability.
Study designs that use exercise sets to task failure (i.e.,
“effort-matched”) could be an alternative and complimen-
tary substitute for volume-matched designs to reduce differ-
ences in muscle activation to objectively evaluate the
anabolic responses due to reduced blood flow. When LL-RE
and high-load resistance exercise (HL-RE) are performed to
task failure, similar protein phosphorylation associated with
increasing muscle protein synthesis is observed 1-h after
exercise, despite different surface EMG characteristics and
external exercise variables such as volume or “time under
tension” (17). Moreover, positive correlations for changes in
phosphorylation of proteins implicated in muscle anabolism
(p70 S6K, rpS6, and ERK-1) and muscle fiber glycogen usage
(to infer fiber activation) are observed (17). These findings
suggest that sufficiently stressing muscle fibers to initiate
signaling cascades implicated in promoting muscle hyper-
trophy is of greater importance than manipulating external
training variables per se. It also supports the use of task fail-
ure study designs to critically evaluate the role BFR may
have on anabolic responses following exercise.
We have previously shown 2-h postexercise muscle mito-
chondrial reactive oxygen species (ROS) emission to be
reduced from baseline only after LL-BFR, possibly as a com-
pensatory response due to the augmented cellular stress or
reduced oxygen availability caused by BFR (18). Since ROS is
a potential signal in the adaptive cell response (19), different
cell signaling might exist when training with BFR compared
with regular exercise. If different signaling responses exist, it
is possible that the time course or magnitude of muscle
growth may be altered after BFR exercise compared with reg-
ular exercise. Despite this possibility, it remains unknown
whether the signaling responses are altered between LL-RE
and LL-BFR when performed to task failure. Furthermore, it
is unclear if potential changes in these signals manifest as
expected training adaptations if tracked over the course of a
training intervention. Thus, the purpose of this study was to
examine if reduced blood flow during exercise alters the
acute signaling and training-induced muscle hypertrophy
responses when exercise is performed to task failure. If the
previously observed reduced muscle oxygen availability and
mitochondrial ROS emission are involved in initiating sig-
naling responses, we hypothesized that the acute signaling
response would increase with LL-RE and LL-BFR but would
be further augmented with LL-BFR. We hypothesized both
conditions would increase muscle size (20, 21) and explored
if BFR could further increase the growth response.
METHODS
Participant Characteristics
Ten young, healthy males (24 ± 1 yr, 177 ± 1 cm, 78 ± 6 kg)
with lower-body resistance training experience (1 day/wk
for 6 mo) were recruited. Exclusion criteria were no major
injuries (6 mo) or musculoskeletal disease. Participants were
performing recreational levels of lower-body resistance
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exercise before starting the study but were not performing a
structured training program for 1 mo before study com-
mencement. This study was reviewed and approved by the
institution’s research ethics board and all participants pro-
vided written informed consent.
Study Overview
Participants were part of a larger project investigating the
acute and chronic physiological and perceptual effects of LL-
RE and LL-BFR. The findings and in-depth methodological
details from these studies are published elsewhere (18, 21, 22).
A summary of the study design and outcomes used herein
can be found in Fig. 1. In brief, a within-subject unilateral
model was used with each leg randomly assigned to perform
LL-RE and LL-BFR to task failure. Task failure was defined as
the inability to maintain the prescribed cadence (1.75 s eccen-
tric and 1.75 s concentric contractions) with proper technique,
despite strong verbal encouragement. In the acute study, par-
ticipants arrived at the laboratory having refrained from lower
body exercise for at least 72 h, alcohol consumption for 24 h,
and food or caffeine consumption for 12 h. A single baseline
vastus lateralis (VL) muscle biopsy, randomized by leg domi-
nance, was obtained under local anesthesia (2% lidocaine
with epinephrine) using the Bergstro € m technique at approxi-
mately mid-thigh. To reduce participant strain, a single rest-
ing biopsy was taken under the assumption that minimal
biochemical differences exist between legs as previously dis-
cussed (23). Using a Smith machine, participants then per-
formed three sets of single-leg squats to task failure at a load
corresponding to 30% 1-repetition maximum with 100 s of
rest between sets. Half of the participants performed LL-RE
followed by LL-BFR and vice versa. A 5- to 10-min rest period
was used between training bouts in each leg. For the LL-BFR
leg, an 11-cm-wide tourniquet was placed on the upper thigh
as high as possible (above the biopsy site), and the pressure
was set corresponding to 60%–70% of the lowest effective
occlusive pressure (LOP) in the seated position. The LOP is
defined as the lowest pressure required to occlude arterial
blood flow at rest and was determined using the Personalized
Tourniquet System (Delfi, Vancouver, Canada). The tourni-
quet remained inflated until the completion of the third set.
The variable pressure (60%–70%) was due to the discomfort
of two participants during the familiarization of BFR (before
the commencement of the study) when set to 70%. Thus, a
10% reduction in pressure was applied to these participants.
The remainder of participants (n = 8) had the pressure set to
70% LOP. After completing the exercise with each leg, partici-
pants consumed a small granola bar (100 calories; 23% energy
as fat, 72% energy as carbohydrate, and 5% energy as protein),
and remained in the laboratory until a muscle biopsy was
obtained from each leg 2-h postexercise. The exercise volume
(load  repetition) was
50% lower with LL-BFR (18). After 7
days of rest, participants initiated 6 wk of training at a fre-
quency of 3 days/wk. Participants were instructed to avoid
other lower-body aerobic or resistance exercises throughout
the 6 wk. Every participant completed all training sessions
with no adverse responses. Half of the participants performed
LL-RE first in each session followed by LL-BFR and vice versa
with 5–10 min rest period between legs. After each session,
25
g of whey protein (
2.5 g leucine/serving; Canadian Protein,
Windsor, ON, Canada) was provided to the participants. Bi-
 RESISTANCE TRAINING TO TASK FAILURE WITH AND WITHOUT BFR

Figure 1. A: study overview. The small arrows under the training intervention represent each training session during the week with a one-repetition maxi-
mum (1-RM) test occurring bi-weekly. B: low-load resistance exercise with blood flow restriction (LL-BFR) and low-load resistance exercise (LL-RE) proto-
cols using the within-subject design. The leg allocated to LL-BFR had the tourniquet pressure applied throughout the exercise sets and rests until the
end of the third set at a pressure corresponding to 60%–70% of the lowest effective occlusion pressure (LOP). C: summary of outcomes presented in
the current study. Created in part with BioRender.

weekly strength testing was performed and was counted as the
first training session of the corresponding week. Throughout
training, if participants exceeded 30 repetitions on the first set,
the load was increased by 2 kg for the subsequent training ses-
sion. Due to a more rapid onset of task failure, total training
volume throughout the intervention was
33% lower with LL-
BFR (21). Whole muscle thickness of the vastus lateralis (VL)
and rectus femoris (RF) was estimated by B-mode ultrasound
with a linear-array probe (8 L-RS Transducer; 4–13 MHz, Vivid
I, GE Healthcare, Chicago, IL) 2–5 days before starting the
acute study and
2-days after the last training bout. Five days
following the last training session, a resting muscle biopsy was
taken from each leg after all postintervention testing. All bi-
opsy samples were taken
3 cm apart from the middle aspect
of the VL, but the exact location was not explicitly recorded.
Muscle samples were freed from blood or connective tissue
and part of each was rapidly placed into ice-cold preservation
buffer (BIOPS) for mitochondrial bioenergetic analyses, em-
bedded in optimal cutting temperature (OCT) medium in iso-
pentane cooled by liquid nitrogen for immunohistochemistry,
and flash-frozen in liquid nitrogen for Western blotting (18, 21).
Indices of Muscle Hypertrophy: Whole Muscle
Thickness and Muscle Fiber Cross-Sectional Area
Before whole muscle thickness data acquisition, the par-
ticipants were supine for
10 min. During this time, the VL
was located at 50% of the distance between the greater tro-
chanter and lateral epicondyle of the femur; the RF was
located at 50% of the distance between the anterior superior
iliac spine and superior aspect of the patella. Two separate
images were acquired for each site with minimal pressure
applied. The data was analyzed immediately after each
image acquisition.
Muscle fiber cross-sectional area was determined using
standard methods as described previously (21). In brief, mus-
cle samples embedded in OCT medium were cut into 10-lm-
thick sections with a cryostat (Thermo Fisher Scientific,
Mississauga, ON, Canada), maintained at 20 C with sam-
ples from each condition placed on the same slide. Muscle
fiber type was identified using primary antibodies against
myosin heavy chain (MHC) I (BA-F8; 1:20 dilution), MHC
IIA/MHC IIX (SC-71; 1:200 dilution), and MHC IIx (6H1; 1:40
dilution) for 1 h. Similarly, serial sections were used to iden-
tify muscle fiber capillaries [collagen IV (M3F7), 1:50 dilu-
tion], sarcolemma [dystrophin (MANDYS1), 1:100 dilution],
and cell nuclei (DAPI; 30 lM). After overnight incubation
and appropriate washes/secondary antibody incubation,
slides were counterstained for cell nuclei using DAPI for 5
min and then washed three times for 5 min each with 1 
PBS. All primary antibodies were purchased from the
Developmental Studies Hybridoma Bank (University of
Iowa), and secondary antibodies were purchased from
Invitrogen (Burlington, ON, Canada). An Axio Observer Z1
microscope (Carl Zeiss, Jena, Germany) was used to image
each section. Muscle fiber CSA and perimeter were calcu-
lated manually in an unblinded manner by tracing around
each fiber using ImageJ (National Institutes of Health,
Bethesda, MD). Due to technique difficulties as previously
described (21), muscle fiber CSA data was lost for one partici-
pant. Because of the low abundance of IIX fibers in most

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 RESISTANCE TRAINING TO TASK FAILURE WITH AND WITHOUT BFR

participants, fiber types were quantified as type I and II
(both IIA and IIX). Hybrid type I/IIA fibers were also
excluded due to low abundance. No differences were
observed for the number of fibers counted or fiber type
between conditions (21).
Western Blotting
Owing to limits on tissue availability and following the re-
moval of inadequate samples or data loss to technical diffi-
culties, 6–8 samples/group were available for all targets.
Specific details, including the amount of protein loaded for
each target, sample size for each target, as well as primary
and secondary antibody manufacturers and concentrations
used can be found in Table 1. Muscle samples were homoge-
nized with a motorized homogenizer in lysis buffer (1%
Triton-X, 50 mM Tris·HCl, 1 mM EDTA, 1 mM EGTA, 50 mM
NaF, 10 mM sodium b-glycerol phosphate, 5 mM sodium
pyrophosphate, 2 mM DTT, 1 mM PMSF, and 10 lg/mL of
each aprotinin, leupeptin, and pepstatin A, pH 7.5). After ho-
mogenization, samples were centrifuged for 10 min (3,000 g
at 4 C), and the supernatants were recovered. A Bradford
Protein Assay (Bio-Rad) was used to determine protein con-
centrations, and samples were prepared in Laemmli buffer.
Samples were heated (5 min at 90 C), and between 5 and 15
lg of protein per target was loaded on SDS-polyacrylamide
gels (between 7.5% and 12%, 150 V, 1 h) and wet transferred
onto a polyvinylidene difluoride membrane (100 V, 1 h). All
samples for each target were transferred on the same mem-
brane to limit variability. Afterward, the membranes were
blocked in 1xTBS-T (0.1% tween) with 5%–7.5% nonfat skim
milk or bovine serum albumin for 1 h followed by incubation
with the primary antibody overnight. Membranes were then
washed in 1xTBS-T (0.1% tween) for 3  5 min, incubated with
appropriate secondary antibodies for 1 h, and washed once
more in 1xTBS-T (minimum 3  5 min). Targets were visual-
ized via chemiluminescence, and bands were quantified
unblinded using a FluorChem HD imaging system (Alpha
Innotech, Santa Clara). All protein targets are expressed as a
percentage of baseline values (Pre), and phosphorylated pro-
teins were first made relative to the unphosphorylated pro-
tein. The phosphorylated or total isoforms of some protein
kinases could not be reliably measured due to the absence of
bands (e.g., bM or dA isoforms for p-CaMKII and total p38c-
MAPK) or insufficient separation (e.g., c or dD isoforms for p-
CaMKII) in all participants. Thus, in the case of multiple
bands or isoforms being present, arrows in the figures indicate
which bands were quantified. Ponceau staining was used for
each membrane to visualize equal loading.
Statistical Analysis
Data were analyzed in SPSS version 26 (IBM, Armonk,
NY), with alpha set a priori at P  0.05. Visualization of Q-Q
plot and the Shapiro-Wilk test were carried out to test nor-
mality. Repeated-measure ANOVA was performed for
Western blot experiments. When significant findings were
observed, pairwise comparisons using a Bonferroni correc-
tion were applied. Pearson correlation coefficients and
Spearman correlation coefficients were used to examine lin-
ear and monotonic relationships between Western blot sig-
nals and/or changes in indices of muscle hypertrophy.
Muscle fiber CSA and ultrasound-derived whole muscle
thickness of the VL and RF data were obtained from our pre-
vious investigation (21). Muscle fiber CSA frequency distribu-
tion was performed using GraphPad Prism 9 (v. 9.0.1, San
Diego, CA) to bin the number of fibers every 1,000 lm2.
Because of the within-subject design, genetic and environ-
mental factors that may influence the adaptive response to
exercise are controlled between conditions. Thus, any varia-
tion between these two conditions within an individual
could be attributed to measurement error and/or the train-
ing modality. Intraclass correlation coefficients (ICC; two-
way random effects, absolute agreement, single rater/

Table 1. Primary and secondary antibodies used for Western blot analysis
Secondary Antibody* Sample Size
Protein Primary Antibody (Concentration) (Concentration) Protein Loaded, lg per Condition
p-AMPKa (T172) CST–cs2535 (1:1,000) Mouse anti-rabbit (1:2,000) 5 8
Total AMPKa2 CST–cs2757 (1:1,000) Mouse anti-rabbit (1:2,000) 5 8
p-CaMKII (T286) CST–cs12716 (1:1,000) Mouse anti-rabbit (1:1,000) 5 7
Total CaMKII CST–cs3362 (1:1,000) Mouse anti-rabbit (1:1,000) 15 7
p-ERK 1/2 (T202/Y204) CST–cs9101 (1:1,000) Mouse anti-rabbit (1:1,000) 5 8
Total ERK 1/2 CST–cs4695 (1:1,000) Mouse anti-rabbit (1:1,000) 5 8
p-JNK 1/2 (T183/Y185) CST–cs4671 (1:1,000) Mouse anti-rabbit (1:1,000) 5 8
Total JNK 1/2 CST–cs9252 (1:1,000) Mouse anti-rabbit (1:1,000) 5 8
p-p38 MAPK (T180/Y182) CST–cs9211 (1:1,000) Mouse anti-rabbit (1:1,000) 5 8
Total p38 MAPK CST–cs9212 (1:1,000) Mouse anti-rabbit (1:1,000) 5 8
p-AKT (T308) CST–cs9275 (1:1,000) Mouse anti-rabbit (1:1,000) 5 8
p-AKT (S473) CST–cs9271 (1:1,000) Mouse anti-rabbit (1:1,000) 5 8
Total AKT CST–cs4691 (1:1,000) Mouse anti-rabbit (1:2,000) 5 8
p-p70 S6K (T389) CST–cs9234 (1:1,000) Goat anti-rabbit (1:2,000) 15 7
Total p70 S6K CST–cs9234 (1:1,000) Goat anti-rabbit (1:2,000) 10 7
p-4E-BP1 (T37/46) CST–cs2855 (1:1,000) Goat anti-rabbit (1:1,000) 10 7
Total 4E-BP1 CST–cs2855 (1:1,000) Goat anti-rabbit (1:1,000) 10 7
p-eEF2 (T56) CST–cs2331 (1:1,000) Goat anti-rabbit (1:2,000) 5 8
Total eEF2 CST–cs2332 (1:1,000) Goat anti-rabbit (1:2,000) 5 8
3-Nitrotyrosine CC–189542 (1:1,000) Mouse anti-rabbit (1:2,000) 10 7
Acetylated-lysine CST–cs9411 (1:1,000) Mouse anti-rabbit (1:1,000) 7.5 6
CC, Cayman chemical; CST, Cell Signaling Technology. Secondary antibodies were purchased from CST (goat anti-rabbit–cs7074) or
Santa Cruz (mouse anti-rabbit–sc-2357).

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 RESISTANCE TRAINING TO TASK FAILURE WITH AND WITHOUT BFR
measurement) were used to assess the similarity of each out-
come between LL-RE and LL-BFR. ICC values were classified
as poor (0.4), fair to good (0.4–0.75), or excellent (0.75;
24). Where applicable, the effect size was estimated using
Cohen’s d using the standardized mean difference. Data are
presented as means ± standard error in figures.
RESULTS
Acute Skeletal Muscle Signaling following LL-RE and LL-
BFR Performed to Task Failure
Regardless of blood flow, the phosphorylation status of sev-
eral kinases typically implicated or thought of as stress
response signals (AMPK, CaMKII, ERK 1/2, JNK 1/2) or global
posttranslational modifications (acetylated-lysine and nitro-
sylated-tyrosine) were not altered after a single bout of resist-
ance exercise (Fig. 2, all P > 0.2). The phosphorylation status of
several proteins along the canonical AKT-mTOR pathway were
also measured (Fig. 2). Phosphorylated AKT(T308) increased
similarly from baseline after LL-RE and LL-BFR (both
145%,
P < 0.05). Phosphorylated AKT(S473) increased only after LL-
BFR (
145%, P = 0.005), but with no differences between train-
ing conditions (P = 0.9). Although the phosphorylation status
did not change for 4E-BP1(T37/46) (P = 0.96) or eEF2(T56) (P =
0.19), a trend was observed with p70 S6K(T389) (LL-RE:
158%,
LL-BFR: 137%, P = 0.06). Collectively, these data suggest BFR
does not augment the acute signaling responses to a bout of
exercise performed to task failure.
Correlations between AKT-mTOR Signaling Pathway
and Training-Induced Muscle Hypertrophy
Despite the similar acute signaling responses, we next
sought to determine the relationship between acute signal-
ing responses and chronic adaptations. The signaling
response along the AKT-mTOR pathway 2-h after the first
exercise bout was correlated with changes in muscle fiber
CSA after 6 wk of training (Fig. 3). Muscle fiber CSA data was
obtained from our previous investigation (21). No associa-
tions were observed between phosphorylated AKT and
changes in muscle fiber CSA for either modality. Negative
correlations were observed for phosphorylated p70 S6K with
changes in Type II muscle fiber CSA after both LL-RE and
LL-BFR. Similarly, negative correlations were observed
between phosphorylated 4E-BP1 and changes in Type II
muscle fiber CSA after LL-BFR, with trends following LL-RE.
Finally, trends were observed between phosphorylated eEF2
and changes in Type I or average muscle fiber CSA only after
LL-BFR (r  0.723, P  0.066). Taken together, these data
suggest that activation of the canonical AKT-mTOR signal-
ing pathway is not related to the adaptive response to low-
load resistance training, regardless of blood flow.
Individual Signaling and Training-Induced Muscle
Hypertrophy Responses
An advantage of the current experimental model is that it
involves training both legs with different blood flow avail-
ability. Given the unexpected relationships between the
AKT-mTOR signaling pathway and changes in muscle fiber
CSA, we next sought to investigate the influence that genetic
and/or environmental factors can have in the acute signaling
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responses within the same participants (Fig. 4). If genetics
and/or environmental factors were primarily responsible for
adaptive changes, then similar responses would be expected
between legs, irrespective of the LL-RE and LL-BFR condi-
tion, with the data points existing close to the line of agree-
ment. Based on the ICCs obtained, five signals had
statistically significant “fair to excellent” strength similar-
ities (4E-BP1, AKT(T308), acetylated-lysine, ERK 1/2, and JNK
1/2), three signals had trends (P  0.105) for “fair to good”
similarities (AMPK, AKT(S473), and p70 S6K), and four signals
had nonsignificant and “poor” similarities (nitrosylated-ty-
rosine, p38 MAPK, CaMKII, and eEF2). Notably, both CaMKII
and eEF2 phosphorylation had several data points deviating
from the line of agreement suggesting a divergent response
because of the BFR (Fig. 4, K and L).
Given the apparent consistent individual signaling responses,
we also investigated possible similarities in training-induced
muscle hypertrophy responses using changes in individual
muscle fiber CSA and whole muscle VL and RF thickness (Fig.
5). The distribution of muscle fiber CSA was similar after LL-RE
and LL-BFR with no leftward shift compared with baseline, sug-
gesting the training did not overtly promote muscle degenera-
tion-regeneration (Fig. 5, A and B). Based on the ICC values, the
changes in Type I CSA had poor similarity between conditions
and were not related (Fig. 5C). By contrast, statistically signifi-
cant fair to good ICC values were observed for changes in Type
II (Fig. 5D) and average (Type I þ II; Fig. 5E) muscle fiber CSA.
Changes in VL and RF thickness had small-moderate effect
sizes favoring LL-BFR, but these were not statistically signifi-
cant (Fig. 5F; 21). Changes in whole muscle VL thickness had
excellent similarity between conditions (Fig. 5G) but were trend-
ing with changes in RF thickness (Fig. 5H). No correlations were
observed between changes in fiber CSA and VL thickness.
These data suggest that individuals will generally respond con-
sistently when training to task failure regardless of differences
in blood flow.
Although most of the acute and chronic responses appear
similar within an individual, there were variable responses
with CaMKII and eEF2 phosphorylation between LL-BFR
and LL-RE 2-h postexercise (see Fig. 4, K and L), which we
aimed to explore further. Specifically, we used Spearman’s
rank-order correlations to determine the possibility that the
initial changes in CaMKII and eEF2 signaling were related to
the adaptive response in muscle fiber CSA following 6 wk of
training (Fig. 6). Although no relationships existed in the LL-
RE condition, inverse relationships were observed after LL-
BFR between eEF2 phosphorylation and changes in average
muscle fiber CSA (r = 0.786, P = 0.036) as well as CaMKII
phosphorylation and changes in Type I fiber CSA (r =
0.886, P = 0.019). No relationships were observed after LL-
BFR between eEF2 and CaMKII phosphorylation (r = 0.214,
P = 0.645).
DISCUSSION
In the present study, we investigated acute signaling
responses 2-h after LL-RE and LL-BFR performed to task fail-
ure and followed the training-induced muscle hypertrophy
responses within an individual. Despite a different amount of
work, acute signaling and chronic adaptive responses were
generally conserved between LL-RE and LL-BFR. In addition,
1269
 RESISTANCE TRAINING TO TASK FAILURE WITH AND WITHOUT BFR

FR

FR

FR

FR
E

LL E

LL E
MW MW

-R

-R

-R

-R
-B

-B

-B

-B
e

e
e

e
(kDa) (kDa)

LL

LL

LL

LL
Pr

Pr
Pr

Pr
LL

LL
60 –
p-AMPK (T172) 62 – p-AKT (T308)

Total AMPK 62 – p-AKT (S473) 60 –

75 –
Total AKT
60 –
p-CaMKII (T286)

50 – p-p70 S6K (T389) 70 –

75 – Total p70 S6K 70 –

Total CaMKII
50 –
p-4E-BP1 (T37/46) 18 –
p-ERK 1/2 (T202/Y204) 44 –
42 –

Total 4E-BP1 18 –
Total ERK 1/2 44 –
42 –

54 –
p-eEF2 (T56) 95 –
p-JNK 1/2 (T183/Y185) 46 –

95 –
Total eEF2
54 –
75 –
Total JNK 1/2 46 –
3-Nitrotyrosine

p-p38 MAPK (T180/Y182) 40 –

50 –

Total p38 MAPK 40 –

Ac-Lysine

25 –

B 200 p=0.06
Pre LL-RE LL-BFR

150
** *
Arbitrary Units (% of Pre)

100

50

0
2)

6)

8)

3)

9)

6)

)
04

85

82

56

e
17

28

30

47

38

/4

in

in
Y2

Y1

Y1

(T
37
(T

(T

(T

(S

(T

os

ys
2/

3/

0/

(T

F2

-L
20

18

18

yr
T
PK

K
T
KI

Ac
AK
(T

(T

(T

ot
P1
AK

S6

eE
aM
AM

itr
-B
p-
2

p-

p-
0

N
1/

1/
C

AP

p7
p-

4E

3-
p-

p-
M

p-
ER

JN

8
p-

p3
p-

p-

Figure 2. Muscle protein signaling before (Pre) and 2-h after a bout of low-load resistance exercise (LL-RE) or low-load resistance exercise with blood
flow restriction (LL-BFR) performed to task failure. A: representative Western blot images for each protein for two participants. B: quantification of signals
expressed relative to Pre-values. Phosphorylated signals were made relative to total protein content. In the case of multiple banding, arrows indicate
which bands were quantified. P < 0.05 vs. Pre. Data are expressed as the group mean with error bars representing the standard error (n = 6–8/group).

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 RESISTANCE TRAINING TO TASK FAILURE WITH AND WITHOUT BFR

A E LL-BFR
1.0
Type I 0.201 -0.039 -0.771 -0.742 0.490 0.439 0.365 -0.614 -0.451 -0.723
Muscle fiber CSA

p=0.666 p=0.934 p=0.073 p=0.091 p=0.265 p=0.324 p=0.421 p=0.195 p=0.369 p=0.066 0.5
from Pre

Type II 0.176 0.009 -0.852 -0.781 0.515 0.579 0.674 -0.876 -0.818 -0.431 0
p=0.706 p=0.984 p=0.031 p=0.067 p=0.237 p=0.174 p=0.097 p=0.022 p=0.047 p=0.334

-0.5
Type I+II 0.264 0.084 -0.807 -0.708 0.425 0.500 0.485 -0.745 -0.623 -0.744
p=0.567 p=0.858 p=0.052 p=0.116 p=0.342 p=0.253 p=0.270 p=0.090 p=0.186 p=0.055
-1.0
8)

3)

9)

6)

8)

3)

6)

6)
56

89
30

47

38

/4

30

47

/4

5
(T

(T
37

37
(T

(S

(T

(T

(S

(T
(T

(T
F2

F2
T

T
T

K
T
AK

AK
P1
AK

P1
AK
S6

S6
eE

eE
-B

-B
p-

p-
p-

p-

p-

p-
0

0
p7

p7
4E

4E
p-

p-
p-

p-
B LL-RE LL-BFR
3000 3000 3000

Type I+II CSA ( from Pre - μm2)

Type II CSA ( from Pre - μm2)
Type I CSA ( from Pre - μm2)

2000 2000 2000

1000 1000 1000

0 0 0 Figure 3. A: correlations between the per-

-1000 -1000 -1000
cent change in signals along the AKT-mTOR
pathway 2-h after the first exercise bout and
-2000 -2000 -2000
absolute changes in Type I, Type II, or aver-
0 100 200
p-AKT (T308) (% of Pre)
300 0 100 200
p-AKT (T308) (% of Pre)
300 0 100 200
p-AKT (T308) (% of Pre)
300 age (Type I þ II) muscle fiber cross-sectional
area (CSA) after 6-wk of training with low-
3000 3000 3000 load resistance exercise (LL-RE) or low-load
resistance exercise with BFR (LL-BFR). The
Type I+II CSA ( from Pre - μm2)
Type II CSA ( from Pre - μm2)
Type I CSA ( from Pre - μm2)

2000 2000 2000

heat map illustrates all correlations using
1000 1000 1000
Pearson’s r where red indicates a positive
0 0 0 correlation and blue indicates a negative
-1000 -1000 -1000
correlation. The r value and P value are pre-
sented in each square. B: graphical repre-
-2000 -2000 -2000
sentation of correlation data for changes in
0 100 200 300 0 100 200 300 0 100 200 300
p-AKT (S473) (% of Pre) p-AKT (S473) (% of Pre) p-AKT (S473) (% of Pre)
Type I (left column), Type II (middle column),
and average (right column) muscle fiber CSA
3000 3000 3000
to the percent change in signals along the
Type I+II CSA ( from Pre - μm2)
Type II CSA ( from Pre - μm2)
Type I CSA ( from Pre - μm2)

2000 2000 2000 AKT-mTOR pathway. Individual circles repre-

1000 1000 1000 sent a single participant and regression lines
0 0 0
for LL-BFR and LL-RE in gray and black,
respectively.
-1000 -1000 -1000

-2000 -2000 -2000

0 100 200 300 0 100 200 300 0 100 200 300

p-p70 S6K (T389) p-p70 S6K (T389) p-p70 S6K (T389)
(% of Pre) (% of Pre) (% of Pre)

3000 3000 3000

Type I+II CSA ( from Pre - μm2)
Type II CSA ( from Pre - μm2)
Type I CSA ( from Pre - μm2)

2000 2000 2000

1000 1000 1000

0 0 0

-1000 -1000 -1000

-2000 -2000 -2000

0 100 200 300 0 100 200 300 0 100 200 300

p-4E-BP1 (T37/46) (% of Pre) p-4E-BP1 (T37/46) (% of Pre) p-4E-BP1 (T37/46) (% of Pre)

3000 3000 3000

Type I+II CSA ( from Pre - μm2)
Type II CSA ( from Pre - μm2)
Type I CSA ( from Pre - μm2)

2000 2000 2000

1000 1000 1000

0 0 0

-1000 -1000 -1000

-2000 -2000 -2000

0 50 100 150 0 50 100 150 0 50 100 150

p-eEF2 (T56) (% of Pre) p-eEF2 (T56) (% of Pre) p-eEF2 (T56) (% of Pre)

similar training responses within an individual support that support the concept that BFR accelerates the time to task fail-
genetic and/or environmental factors are more important ure and muscular stress with low-loads leading to training-
than BFR in mediating the adaptive responses when contrac- induced muscle hypertrophy but does not support that BFR
tions are performed to task failure. Overall, these results augments the adaptive response.

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 RESISTANCE TRAINING TO TASK FAILURE WITH AND WITHOUT BFR

A 300 p-4E-BP1(T37/46)
B 300 p-AKT (T308)
C 300 Ac-Lysine
D 300 p-ERK 1/2 (T202/Y204)

250 250 250 250

LL-BFR (% of Pre)

LL-BFR (% of Pre)

LL-BFR (% of Pre)

LL-BFR (% of Pre)
200 200 200 200

150 150 150 150

100 100 100 100

50 50 50 50

0 r = 0.983, p < 0.0001 0 r = 0.879, p = 0.004 0 r = 0.850, p = 0.03 0 r = 0.682, p = 0.06

ICC = 0.983, p < 0.0001 ICC = 0.889, p = 0.001 ICC = 0.869, p = 0.008 ICC = 0.695, p = 0.023
0 50 100 150 200 250 300 0 50 100 150 200 250 300 0 50 100 150 200 250 300 0 50 100 150 200 250 300
LL-RE (% of Pre) LL-RE (% of Pre) LL-RE (% of Pre) LL-RE (% of Pre)

E 300 p-JNK 1/2 (T183/Y185)

F 300 p-AMPK (T172)
G 300 p-AKT (S473)
H 300 p-P70 S6K(T389)

250 250 250 250

LL-BFR (% of Pre)

LL-BFR (% of Pre)

LL-BFR (% of Pre)

LL-BFR (% of Pre)
200 200 200 200

150 150 150 150

100 100 100 100

50 50 50 50

0 r = 0.742, p = 0.035 0 r = 0.584, p = 0.13 0 r = 0.654, p = 0.08 0 r = 0.501, p = 0.25

ICC = 0.684, p = 0.025 ICC = 0.578, p = 0.057 ICC = 0.519, p = 0.074
7 ICC = 0.514, p = 0.105
0 50 100 150 200 250 300 0 50 100 150 200 250 300 0 50 100 150 200 250 300 0 50 100 150 200 250 300
LL-RE (% of Pre) LL-RE (% of Pre) LL-RE (% of Pre) LL-RE (% of Pre)

I 300 3-Nitrotyrosine
J 300 p-p38 MAPK (T180/Y182)
K 300 p-CaMKII (T286)
L 300 p-eEF2 (T56)

250 250 250 250

LL-BFR (% of Pre)

LL-BFR (% of Pre)

LL-BFR (% of Pre)

LL-BFR (% of Pre)
200 200 200 200

150 150 150 150

100 100 100 100

50 50 50 50

0 r = 0.135, p = 0.8 0 r = -0.454, p = 0.26 0 r = -0.523, p = 0.23 0 r = -0.126, p = 0.8

ICC = 0.125, p = 0.4 ICC = -0.328, p = 0.78 ICC = -0.555, p = 0.9 ICC = -0.093, p = 0.63
0 50 100 150 200 250 300 0 50 100 150 200 250 300 0 50 100 150 200 250 300 0 50 100 150 200 250 300
LL-RE (% of Pre) LL-RE (% of Pre) LL-RE (% of Pre) LL-RE (% of Pre)

Figure 4. Individual data points for the percent change in signals 2-h after a bout of low-load resistance exercise (LL-RE) or low-load resistance exercise
with blood flow restriction (LL-BFR) performed to task failure (A-L). Specific signals are indicated in the top left of the graph with the dashed line repre-
senting the line of agreement. If the individual circle exists on the left side of the line, this suggests a greater change following LL-BFR compared with LL-
RE within that individual and vice versa if on the right side of the line. Pearson’s r correlation coefficient and intraclass correlation coefficient (ICC) with
their respective P values are presented in the bottom left of each graph. n = 6–8/target.

Training adaptations are the result of repetitive activation of differences in muscle activation (31), but with different muscle
signaling pathways leading to changes in gene transcription oxygen availability to evaluate the role that additional meta-
and translation (19). Previous work has supported the notion bolic stress may have on hallmark signaling pathways.
that BFR during resistance exercise increases several signaling Although the phosphorylation status of kinases (AMPK,
pathways that are associated with a broad spectrum of training CaMKII, ERK 1/2, JNK 1/2, and p38 MAPK) and global post-
adaptations (5–9, 25). However, work comparing LL-BFR to LL- translational modifications (nitrosylated-tyrosine and acety-
RE has typically matched conditions to the same number of lated-lysine) often associated as exercise or stress response
repetitions per set or “volume-matched.” As a result, volume- sensors were not altered after exercise, protein phosphoryla-
matched designs likely lead to greater muscle activation (12, 13) tion along the AKT-mTOR pathway was increased similarly
and metabolic stress (26, 27) when exercise is performed with following both LL-BFR and LL-RE. The activation of the
reduced blood flow. When muscle activation is controlled for AKT-mTOR pathway after resistance exercise has been sup-
such as during electrically stimulated (28, 29) or maximal vol- ported previously regardless of reduced blood flow (32), an
untary (30) contractions, reduced blood flow still increases observation that is attenuated with an mTOR inhibitor, rapa-
markers indicative of increased metabolic stress (e.g., increased mycin resulting in blunted muscle protein synthesis rates (8,
muscle lactate, AMP, and ADP concentrations). Furthermore, 33). However, our findings suggest LL-RE activates this path-
we have previously shown that estimated muscle oxygenation way to a similar magnitude as LL-BFR after exercise, as sup-
(as inferred by near-infrared spectroscopy) is reduced during ported by high ICC values with AKT(T308) phosphorylation
LL-BFR compared with LL-RE despite both performing exer- and trends with AKT(S473) and p70 S6K phosphorylation
cise to task failure (18). Given this, we sought to investigate the between conditions. Of note, no positive associations were
acute signaling responses after contractions performed to task observed between the phosphorylation status of these pro-
failure with and without BFR as a model to reduce potential teins 2-h after the first bout of exercise with changes in

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 RESISTANCE TRAINING TO TASK FAILURE WITH AND WITHOUT BFR

A B LL-RE LL-BFR
Type I Type II Type I+II
Cohen’s d = 0.23 Cohen’s d = 0.008 Cohen’s d = 0.049
3000
25 Pre 2500
LL-RE
2000

Muscle Fiber CSA (∆ from Pre - μm2)

20 LL-BFR
1500
Distribution (%)

15 1000

500
10
0

5 -500

-1000
0
-1500
0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 11000 12000 13000 14000
-2000
Bin for Fiber CSA (μm2)
-2500

C D E

LL-BFR - Type I+II CSA (∆ from Pre - μm2)

3000
LL-BFR - Type II CSA (∆ from Pre - μm2)

3000 3000
LL-BFR - Type I CSA (∆ from Pre - μm2)

2000 2000 2000

1000 1000 1000

0 0 0

-1000 -1000 -1000

-2000 r = 0.279, p = 0.48 -2000 r = 0.563, p = 0.115 -2000 r = 0.623, p = 0.073

ICC = 0.259, p = 0.25 ICC = 0.579, p = 0.05 ICC = 0.637, p = 0.031
-2000 -1000 0 1000 2000 3000 -2000 -1000 0 1000 2000 3000 -2000 -1000 0 1000 2000 3000
LL-RE - Type I CSA (∆ from Pre - μm2) LL-RE - Type II CSA (∆ from Pre - μm2) LL-RE - Type I+II CSA (∆ from Pre - μm2)

F LL-RE LL-BFR G 0.8 H 0.8

LL-BFR - RF Muscle Thickness
LL-BFR - VL Muscle Thickness

VL Thickness RF Thickness 0.6 0.6

Whole-Muscle Thickness (∆ from Pre - cm)

Cohen’s d = 0.43 Cohen’s d = 0.59

0.8
(∆ from Pre - cm)

(∆ from Pre - cm)

0.7 0.4 0.4

0.6
0.5 0.2 0.2
0.4
0.3 0.0 0.0
0.2
0.1 -0.2 -0.2
0.0
-0.1 -0.4 r = 0.646, p = 0.044 -0.4 r = 0.478, p = 0.162
-0.2 ICC = 0.770, p = 0.016 ICC = 0.421 p = 0.069
-0.3 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8
-0.4
LL-RE - VL Muscle Thickness LL-RE - RF Muscle Thickness
(∆ from Pre - cm) (∆ from Pre - cm)

Figure 5. A: frequency of Type I þ II muscle fiber cross-sectional area (CSA) before (Pre; dotted line) and after 6-wk of training with low-load resistance
exercise (LL-RE; black dashed lined) or low-load resistance exercise with blood flow restriction (LL-BFR; gray dashed line). The number of fibers were
binned every 1,000 lm2. B: change in Type I, Type II, or average (Type I þ II) muscle fiber CSA from baseline after 6-wk of training with LL-RE (black bars)
and LL-BFR (gray bars). C–E: graphical representation of each participant illustrating changes in Type I, Type II, or average (Type I þ II) muscle fiber CSA
between conditions. F: changes in whole muscle vastus lateralis (VL) and rectus femoris (RF) thickness. G and H: graphical representation of each partici-
pant illustrating changes in VL and RF muscle thickness between conditions. Individual data is expressed as circles and bars as group means with the
standard error (n = 9–10/group). Effect size between conditions was provided using Cohen’s d, the dashed lines are the line of agreement and Pearson’s
r correlation coefficient as well as intraclass correlation coefficient (ICC) with P values are presented at the bottom of each graph, where applicable.

muscle fiber CSA over 6 wk of training. Instead, inverse cor- lowest training-induced muscle hypertrophy. This observa-
relations were observed between signaling proteins down- tion is in stark contrast with previous work demonstrating a
stream of mTOR (i.e., p70 S6K and 4E-BP1) with changes in strong positive correlation (r = 0.82) between changes in
Type II muscle fiber CSA. These findings suggest those who Type IIA muscle fiber CSA after 14 wk of training with
had the greatest signaling response (and presumably poten- changes in p70 S6K phosphorylation 30-min after the first
tial to increase muscle protein synthesis rates) had the bout of resistance exercise in untrained participants (34). It

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 RESISTANCE TRAINING TO TASK FAILURE WITH AND WITHOUT BFR

A 3000 B 3000 C 3000

Type I+II CSA ( from Pre - μm2)

Type II CSA ( from Pre - μm2)
Type I CSA ( from Pre - μm2)

2000 2000 2000

1000 1000 1000

0 0 0

-1000 -1000 -1000

-2000 -2000 -2000

= 0.200, p = 0.704 = 0.200, p = 0.704 = 0.200, p = 0.704
0 50 100 150 200 250 0 50 100 150 200 250 0 50 100 150 200 250
p-CaMKII (T286) (% of Pre) p-CaMKII (T286) (% of Pre) p-CaMKII (T286) (% of Pre)
LL-RE

D 3000 E 3000 F 3000

Type I+II CSA ( from Pre - μm2)

Type II CSA ( from Pre - μm2)
Type I CSA ( from Pre - μm2)

2000 2000 2000

1000 1000 1000

0 0 0

-1000 -1000 -1000

-2000 -2000 -2000

= 0.286, p = 0.535 = 0.393, p = 0.383 = 0.393, p = 0.383
0 50 100 150 0 50 100 150 0 50 100 150
p-eEF2 (T56) (% of Pre) p-eEF2 (T56) (% of Pre) p-eEF2 (T56) (% of Pre)

G 3000
H 3000 I
Type I+II CSA ( from Pre - μm2) 3000
Type II CSA ( from Pre - μm2)
Type I CSA ( from Pre - μm2)

2000 2000 2000

1000 1000 1000

0 0 0

-1000 -1000 -1000

-2000 -2000 -2000

= -0.536, p = 0.215 = -0.357, p = 0.432 = -0.786, p = 0.036

0 50 100 150 0 50 100 150 0 50 100 150

LL-BFR

p-eEF2 (T56) (% of Pre) p-eEF2 (T56) (% of Pre) p-eEF2 (T56) (% of Pre)

J 3000 K 3000 L 3000

Type I+II CSA ( from Pre - μm2)
Type II CSA ( from Pre - μm2)
Type I CSA ( from Pre - μm2)

2000 2000 2000

1000 1000 1000

0 0 0

-1000 -1000 -1000

-2000 -2000 -2000

= -0.886, p = 0.019 = -0.714, p = 0.111 = -0.771, p = 0.072

0 100 200 300 400 0 100 200 300 400 0 100 200 300 400
p-CaMKII (T286) (% of Pre) p-CaMKII (T286) (% of Pre) p-CaMKII (T286) (% of Pre)

Figure 6. Correlation analysis between changes in Type I, II, or average (Type I þ II) muscle fiber CSA with training and changes in e-EF2 or CaMKII phos-
phorylation 2-h after the first exercise bout with low-load resistance exercise (LL-RE; A–F) or low-load resistance exercise with blood flow restriction (LL-
BFR; G–L). Spearman’s correlation coefficient and P value are shown in the bottom left of each graph. n = 6–7/group.

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 RESISTANCE TRAINING TO TASK FAILURE WITH AND WITHOUT BFR
is difficult to explain these discrepancies, but they do not
seem likely to be explained by differences in training status
between the study of Terzis et al. (34) and the present study
(i.e., untrained vs. trained). This is because the increased
myofibrillar protein synthesis rates at the onset of HL-RE
training in individuals unaccustomed to resistance exercise
training for at least 3–6 mo (i.e., exercise-naïve) also do not
correlate with training-induced muscle hypertrophy (35, 36).
It has been hypothesized that part of the increased protein
synthesis response at the onset of training in the exercise-
naïve state is related to repairing and replacing damaged
proteins. After this initial period, the increased protein syn-
thesis rates are primarily dedicated to increasing muscle
fiber size (36). Considering this, we purposefully recruited
individuals who were accustomed to resistance exercise
training to reduce the “exercise-naïve” signaling response
that could occur with that population. Nonetheless, it is pos-
sible a similar muscle damage-repair response occurred in
some of our participants at the onset of LL-RE and LL-BFR
training and could be a future avenue of research.
Leveraging our within-subject design, we tested if individ-
ual responses within a participant were similar across LL-RE
and LL-BFR training. High ICC values would suggest genetic
and/or environmental factors play a more influential role in
mediating the training response as opposed to factors related
to the intentional reduction in blood flow. Changes in Type
II and average (i.e., both Type I and II) muscle fiber CSA and
VL muscle thickness were similar within the same individual
as indicated by fair to excellent ICC values. That is, both legs
had similar changes at the microscopic and macroscopic lev-
els in the same muscle. Although we cannot explain the
remaining variability in our analyses (i.e., measurement
error vs. exercise modality), these results generally support
that LL-RE and LL-BFR are both capable of stimulating simi-
lar muscle growth in the same individual. Notably, these
findings were observed despite the LL-BFR condition accu-
mulating a lower amount of total exercise volume through-
out training [
33% less vs. LL-RE (21)]. This would support
the concept that BFR accelerates the motor unit recruitment
and consequently activation of similar anabolic signaling
pathways as resistance exercise with intact blood flow.
However, we further explored if the reduced muscle oxygen
availability [as estimated in our previous work (18)] was able
to influence different underlying signaling pathways related
to muscle anabolism. To explore this further, we examined
the phosphorylation status of eEF2 and CaMKII 2-h postexer-
cise to changes in muscle fiber hypertrophy. These proteins
were selected as we observed a disproportionate change in
eEF2 and CaMKII phosphorylation in one limb/condition
versus the other for several participants (see Fig. 4, K and L).
Our analysis showed an inverse relationship with the change
in average muscle fiber CSA and eEF2 phosphorylation [a
lower phosphorylation promotes elongation during protein
translation (37)] as well as between CaMKII phosphorylation
and the change in Type I muscle fiber CSA only after LL-BFR
(Fig. 6). Speculatively, the signaling pathways that these pro-
teins are associated with may be regulated differentially due
to reduced muscle oxygen availability as opposed to the gen-
eral effects of exercise. But this speculation requires caution
as the comparisons were made with relatively small sample
sizes (n = 6–7/group).
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Methodological Considerations and Limitations
Although most of our findings suggest the acute and
chronic responses were similar at low loads irrespective of
blood flow, there are some considerations and limitations in
this study. First, the acute signaling was obtained 2-h after
exercise, thus we cannot rule out differences before and after
the selected timepoint between the two conditions, particu-
larly since some signals may rapidly dephosphorylate soon af-
ter exercise [e.g., AMPK or the family of MAPKs; (38–40)] or
increase for over 24 h after exercise (e.g., p70 S6K; 8).
Moreover, it is possible that the discrepancy between the cor-
relation of phosphorylated p70 S6K and change in Type II CSA
in the present study with the study of Terzis et al. (34) could
be due to greater muscle damage after the first bout in some of
our participants causing an increased signaling response to
repair the damaged tissue. To reduce the possibility of exer-
cise-induced muscle damage, resistance-trained individuals
were recruited, but these discrepancies support that caution
should be taken when inferring changes in signaling pathways
in the early recovery period with the expected hypertrophic
training response. Second, because participants trained each
leg in close proximity (
5–10 min apart), blood-borne factor(s)
may have influenced the present findings as previous work
has shown that LL-BFR may further increase the muscle
growth response with regular exercise (41). However, this has
not been replicated (42) and previous work supports that natu-
rally increasing blood-borne factors (e.g., anabolic hormones)
with exercise does not further increase muscle signaling path-
ways, protein synthesis, and growth responses with HL-RE
training (43, 44). Third, we estimated and compared muscle
growth responses using microscopic (muscle fiber CSA) and
macroscopic (ultrasound-derived muscle thickness) methods
and no relationship was observed between these outcomes.
Although we are not the first to demonstrate a dissociation
between these indices of muscle hypertrophy [e.g., Ref. (45)],
this could be partially explained by the appreciable variability
associated with repeat small biopsy samples for muscle fiber
area [i.e., coefficient of variation ranging between
10% and
20% (46–48)], thus, caution is required when interpreting this
dissociation. In addition, the small-moderate effect sizes for
changes in VL and RF muscle-thickness in favor of LL-BFR
training could be interpreted as BFR inducing prolonged mus-
cle edema due to muscle damage. However, several indirect
lines of evidence suggest limited muscle-damage-induced
edema in our study. For example, the functional tests (e.g., 1-
RM and muscular endurance task) improved with training in
our cohort (21) and no leftward shift in the distribution of mus-
cle fiber CSA was observed (see Fig. 5A). Exploratory analysis
of the whole muscle ultrasound images also demonstrated a
reduction in echo intensity in both groups (Supplemental Fig.
S1; all Supplemental material is available at https://doi.org/
10.6084/m9.figshare.22540222), which is in contrast to a
recent study that showed evidence of muscle fiber damage
and regeneration with strenuous, high-frequency LL-BFR
training (49).
Conclusion
The present findings suggest the addition of BFR during
resistance exercise reduces the required exercise volume to
induce similar acute signaling and training-induced muscle
1275
 RESISTANCE TRAINING TO TASK FAILURE WITH AND WITHOUT BFR
hypertrophy responses. They also indicate genetic and/or
environmental factors are more critical to cause an adaptive
response than the intentional BFR. Overall, BFR likely accel-
erates the recruitment of large motor units and conse-
quently, accelerates the stress placed on individual fibers to
initiate signaling pathways causing an adaptive muscle
growth response.
DATA AVAILABILITY
Data will be made available upon reasonable request.
SUPPLEMENTAL DATA
Supplemental Fig. S1: https://doi.org/10.6084/m9.figshare.
22540222.
ACKNOWLEDGMENTS
The figures were in part created with BioRender.com and are
published with permission.
GRANTS
This work was supported by a Discovery Grant from the
Natural Sciences and Engineering Research Council of Canada
(03974 to J.F.B.), the Canadian Foundation for Innovation (35460
to J.F.B.), and the Ontario Ministry of Research, Innovation, and
Science (460597 to J.F.B.).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by
the authors.
AUTHOR CONTRIBUTIONS
C.P., G.P.H., and J.F.B. conceived and designed research; C.P.
performed experiments; C.P. analyzed data; C.P., G.P.H., and J.F.B.
interpreted results of experiments; C.P. prepared figures; C.P.
drafted manuscript; C.P., G.P.H., and J.F.B. edited and revised
manuscript; C.P., G.P.H., and J.F.B. approved final version of
manuscript.
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