Calf Muscle Volume Estimates: Implications
for Botulinum Toxin Treatment?
Thomas Bandholm, PT, MSc*†, Stig Sonne-Holm, MD, DSc*, Carsten Thomsen, MD, DSc‡,
Jesper Bencke, MSc, PhD*, Søren A. Pedersen, MD*§, and Bente R. Jensen, MSc, PhD¶
An optimal botulinum toxin dose may be related to the
volume of the targeted muscle. We investigated the
suitability of using ultrasound and anthropometry to
estimate gastrocnemius and soleus muscle volume.
Gastrocnemius and soleus muscle thickness was mea-
sured in 11 cadaveric human legs, using ultrasound.
Lower leg length was tape-measured. Muscle volume
was determined by water displacement of the dissected
muscles. Simple and multiple regression analyses, us-
ing muscle thickness and lower leg length as indepen-
dent variables, were performed to establish muscle
volume prediction equations from the muscle volume
measured by water displacement. Validating the equa-
tions based on simple regression analyses resulted in a
correlation (r2) of 0.373 and 0.518 (P < 0.047), and a
standard error of the estimate of 24.4 cm3 (11.7% of
the measured mean muscle volume) and 33.2 cm3
(12.7%) for the gastrocnemius and soleus muscles,
respectively. The corresponding values for the mul-
tiple regression analyses were an r2 of 0.497 and
0.650 (P < 0.017), and a standard error of the
estimate of 21.9 cm3 (10.5%) and 28.4 cm3 (10.8%)
for the gastrocnemius and soleus muscles, respec-
tively. It seems possible to estimate the volume of
individual plantar flexor muscles using ultrasound
and anthropometry. This possibility should be inves-
tigated further in living humans. © 2007 by
Elsevier Inc. All rights reserved.
Bandholm T, Sonne-Holm S, Thomsen C, Bencke J,
Pedersen SA, Jensen BR. Calf muscle volume esti-
mates: Implications for botulinum toxin treatment?
Pediatr Neurol 2007;37:263-269.
From the *Gait Analysis Laboratory, Department of Orthopedic
Surgery, and †Department of Physical Therapy, Hvidovre University
Hospital, Copenhagen, ‡Department of Radiology X 3023,
Rigshospitalet, Copenhagen, §Department of Pediatrics, Hvidovre
University Hospital, Copenhagen, and ¶Department of Exercise and
Sport Sciences, University of Copenhagen, Copenhagen, Denmark.
© 2007 by Elsevier Inc. All rights reserved.
doi:10.1016/j.pediatrneurol.2007.05.019 ● 0887-8994/07/$—see front matter
Introduction
Muscle volume estimates are useful in the assessment of
muscle function in terms of maximal force-generating
capabilities. In fact, muscle volume seems to be more
closely related to maximal muscle torque, compared with
muscle cross-sectional area [1,2]. From a pharmacologic
point of view, quantification of muscle volume might
improve the dosing standards of botulinum toxin type A
(BTA) when administered as intramuscular injections, to
temporarily reduce focal-muscle spasticity.
Even though the pharmacokinetics of BTA are not fully
understood, there are indications that the clinical outcome
of treatment with BTA in children with cerebral palsy is
related to the injected dose [3,4]. The current BTA
treatment recommendations for children with cerebral
palsy suggest that the BTA dosage for each muscle should
be calculated from the total body weight of the patient [5].
It is possible, however, that determining optimal dosing
standards in cerebral palsy may require an estimate of the
volume of the muscle targeted for injections with BTA.
For example, two children of equal body weight with
cerebral palsy may not necessarily demonstrate a similar
volume of a specific skeletal muscle targeted for intramus-
cular injections with BTA. Assuming a similar diffusion of
BTA within the tissue, the average intramuscular concen-
tration of the toxin will likely differ between the two
children if dosage is based exclusively on body weight.
Hence, the degree of spasticity reduction caused by the
toxin will likely differ between the two children as well.
In children with cerebral palsy, the muscles of the calf
are often hyperactive because of spasticity [6,7], and many
children with cerebral palsy manifest spastic equinus of
the foot [8]. Therefore, studies aiming to investigate if the
volume of calf muscles injected with BTA is related to
treatment outcome will require the volume of these mus-
Communications should be addressed to:
T. Bandholm; Gait Analysis Laboratory (Section 247), Department of
Orthopedic Surgery; Hvidovre University Hospital; Kettegaard Allé
30; DK-2650 Hvidovre, Denmark.
E-mail: thomas.bandholm@hvh.regionh.dk
Received March 12, 2007; accepted May 30, 2007.
Bandholm et al: Muscle Volume Estimates 263
cles to be quantified in a quick, low-cost manner. Further-
more, as objective measurements of spasticity using iso-
kinetic dynamometry are emerging [6,7,9-11], the degree
of spasticity reduction could be assessed objectively in
muscle volume-dose studies.
Previous attempts were made to estimate arm and leg
muscle group volume and muscle mass based on anthro-
pometry [12,13]. Others combined anthropometric and
ultrasound measurements to establish prediction equations
for limb muscle volume [14]. In this way, muscle-volume
prediction equations of the elbow flexor and extensor
[1,15], quadriceps femoris [16], and tibialis anterior [17]
muscles were developed. For the plantar flexors specifi-
cally, Miyatani et al. established a muscle-volume predic-
tion equation for the plantar flexor muscle group, using
muscle group thickness and lower leg length as indepen-
dent variables [14]. To our knowledge, the accuracy of
estimating the volume of individual plantar flexor muscles
using ultrasound has not been addressed. Furthermore,
muscle-volume prediction equations have not usually been
validated against the true muscle volume measured as the
amount of displaced water when the muscle is lowered
into a water-filled cylinder, because this technique can be Figure 1. Measurement sites used for ultrasound measurements (top)
and an example of an ultrasound scan (bottom). mGA, measurement site
performed in cadavers only. for the medial gastrocnemius muscle; IGA, measurement site for the
The primary objective of the present study was to lateral gastrocnemius muscle; SOL, measurement site for the soleus
muscle; GA, gastrocnemius; SOL, soleus.
investigate the suitability of using ultrasonography mus-
cle-thickness measurements and anthropometry to esti-
mate gastrocnemius and soleus muscle volumes in cadav- Lines connecting the MTJs of the medial and lateral gastrocnemius head
eric human legs. We tested the hypothesis that the volume (mGA and IGA, respectively), and the insertion of the Achilles tendon
of gastrocnemius and soleus muscles can be estimated with the corresponding 25%, 75%, and 50% marks indicated on the
posterior circumferential knee epicondylar line, were then drawn on the
using muscle thickness and anthropometric measurements.
skin, and distances were measured (Fig 1). Half of the measured distance
was indicated on the skin for each line, leaving one measurement site for
each of the gastrocnemius muscle heads and one measurement site for the
Methods
soleus muscle (SOL) at approximately 50% of the muscle-belly length.
The subcutaneous adipose tissue-muscle to muscle-fascia (mGA and
Materials IGA) and muscle-fascia to muscle-fascia (SOL) interface distances
(muscle thickness) were then determined from the ultrasound image (Fig 1).
Eleven whole, frozen, cadaveric human legs were used after thawing Muscle thickness was determined for the mGA, IGA, and SOL as a mean
overnight. No information could be obtained concerning age, sex, body of three scans for each measurement site. The same investigator per-
weight, and cause of death of the deceased because of medical-school formed all ultrasound measurements, and the mean difference of the three
routines. recordings at each measurement site was ⬍0.4 mm. Gastrocnemius
muscle thickness was expressed as a mean of the mGA and IGA muscle
thickness. During scans, great care was taken not to compress the dermal
Muscle Thickness Measured by Ultrasound surface.

The legs were placed in prone position (patella pointing downward

into the table) and supported above the knee joint. The ankle joint angle
was approximately 100 degrees (neutral position). Lower leg length was
determined as the distance from the lateral knee-joint line to the most
prominent part of the lateral malleolus. The posterior circumferential
knee epicondylar distance was measured as the length of the drawn line
on the skin connecting the medial and lateral femoral epicondyles (Fig 1).
Subsequently, distances corresponding to 25%, 50%, and 75% of the
posterior circumferential knee epicondylar distance were marked on the
skin.
Ultrasound scans were performed using a Diasonics VST Master
Series ultrasound scanner (scanning frequency, 5 MHz) (GE Healthcare).
With the transmission gel-coated transducer oriented in the axial plane
and perpendicular to the muscle of interest, the insertion of the Achilles
tendon onto the calcaneus and the distal myo-tendinous junction (MTJ,
most distal point) for each of the two heads of the gastrocnemius muscle
were determined and indicated on the skin with a permanent marker.
Muscle Volume Measured by Water Displacement
All 11 legs were dissected. By means of a long incision from the
poples to the heel, the two muscles were isolated, taking away the fascia
surrounding the compartment. The lateral and medial heads of the
gastrocnemius muscle were taken down from the condyles of the femur,
and the soleus muscle from the posterior surface of the fibula, the
tendinous arch, and the posterior surface of the tibia. The space between
the deep flexor compartment and the gastrocnemius and soleus muscles
was opened by blunt dissection. Muscles were detached cutting the
Achilles tendon distally. The tibial nerve and popliteal vessels, as well as
the plantaris muscle, were detached. The muscles were separated, and all
tendinious and fatty tissue was removed. The volume of each muscle was
then measured as the volume of displaced (0.9%, isotonic) salt water
when the muscle was fully submerged in a salt water-filled measuring
cylinder (600-mm diameter, 1000-mL volume, Bie & Berntsen, Rødovre,

264 PEDIATRIC NEUROLOGY Vol. 37 No. 4

Denmark). The diameter of the measuring cylinder was the smallest
possible that could be applied while still obtaining accurate measure-
ments. This was performed three times for each muscle by the same
investigator, and muscle volume was expressed as a mean of the three
recordings. The mean difference of the three muscle-volume recordings
was ⬍3 cm3 for both muscles.
Data Analyses
Simple and multiple regression analyses were performed, using the
muscle volume measured by water displacement as the dependent
variable for both muscles. The simple regression analyses used [␲ ⫻
(muscle thickness/2)2 ⫻ lower leg length] as the independent variable,
and the multiple regression analyses used muscle thickness and lower leg
length as the independent variables for both muscles, respectively [16].
We also investigated if using the mean gastrocnemius muscle length (the
distance from the posterior circumferential knee epicondylar line to the
mean mGA and IGA MTJ) as the independent variable instead of lower
leg length produced better results. Because this was not the case, we
chose to use lower leg length as the anthropometric measurement to
facilitate subsequent between-study comparisons. Using the established
muscle-volume prediction equations, a muscle volume for the gastroc-
nemius and soleus muscles was calculated for each leg. To assess the
criterion validity of the equations in predicting the measured muscle
volume, the standard error of the estimate (SEE) and validity correlation
(r2) were calculated, using the measured muscle volume as the criterion.
The SEE was calculated using the equation SDdiff /公2, where SDdiff is
the standard deviation of the difference scores between the estimated and
measured muscle volume. The SEEs were expressed as absolute values
and values relative to the measured muscle volume [14]. Paired-samples
t tests were used to examine if systematic differences existed between the
estimated and measured muscle volume. To examine if the differences
between the estimated and measured muscle volume were related to the
size of the measured muscle volume (heteroscedasticity), the muscle-
volume difference (measured ⫺ estimated muscle volume) was plotted
against the mean of the measured and estimated muscle volume, with
corresponding limits of agreement (mean difference ⫾ 2 SD [18]).
Moreover, Pearson correlation coefficients were calculated to quan-
tify the degree of relationship between the numerical muscle-volume
differences and muscle-volume means, as indicated in Bland and
Altman [18] plots. The variability around mean values is expressed as
⫾1 SD, unless otherwise stated. The level of significance was set at
P ⱕ 0.05.
Results
Measured Anthropometry, Muscle Volume,
and Thickness
The measured muscle volume was 208.5 ⫾ 43.6 cm3
and 262.1 ⫾ 67.7 cm3 for the gastrocnemius and soleus
muscles, respectively. Muscle thickness was 15.8 ⫾ 2.8
mm, 12.4 ⫾ 3.3 mm, and 18.7 ⫾ 4.7 mm for mGA, IGA,
and SOL, respectively. Leg length was 38.5 ⫾ 3.9 cm, and
muscle length was 23.0 ⫾ 3.7 cm and 22.3 ⫾ 4.7 cm for
mGA and IGA, respectively.
Simple Regression Analyses
In simple regression analyses, [␲ ⫻ (muscle thickness/
2)2 ⫻ lower leg length] was significantly correlated with
the measured muscle volume for the gastrocnemius (r ⫽
0.656, P ⫽ 0.028) and soleus (r ⫽ 0.720, P ⫽ 0.012)
muscles. The muscle-volume prediction equations based on
simple regression analyses are given in Table 1. Using these
equations to estimate muscle volume resulted in an r2 and
SEE of 0.373 (P ⫽ 0.046) and 24.4 cm3 (11.7%) for the
gastrocnemius muscle, and an r2 and SEE of 0.518 (P ⫽
0.012) and 33.2 cm3 (12.7%) for the soleus muscle when
validated against the measured muscle volume (Fig 2A, B).
No systematic differences existed between the estimated and
measured muscle volume (P ⬎ 0.05), and the Bland and
Altman plots indicated no significant correlation between
muscle-volume differences and the means for any of the two
muscles (r ⬍ 0.209, P ⬎ 0.537, Fig 3A, B).
Multiple Regression Analyses
In multiple regression analyses, muscle thickness and
lower leg length were significant contributors to the
measured muscle volume, and explained 31% and 27%,

Table 1. Regression equations for predicting muscle volume

Equation r2 (P Value) SEE (cm3) (%)

Simple regression
Gastrocnemius y ⫽ 0.987x ⫹ 147.1 0.373 (0.046) 24.4 (11.7)
Soleus y ⫽ 0.238x ⫹ 154.9 0.518 (0.012) 33.2 (12.7)
Multiple regression
Gastrocnemius Y ⫽ 85.3X1 ⫹ 5.0X2 ⫺ 104.9 0.497 (0.016) 21.9 (10.5)
Soleus Y ⫽ 73.6X1 ⫹ 10.6X2 ⫺ 284.4 0.650 (0.003) 28.4 (10.8)

Abbreviations:
LL ⫽ Lower leg length
MT ⫽ Muscle thickness
r2 ⫽ Validity correlation between estimated and measured muscle volume
SEE ⫽ Standard error of the estimate
x ⫽ ␲ ⫻ (MT/2)2 ⫻ LL
X1 ⫽ Muscle thickness (cm)
X2 ⫽ Lower leg length (cm)
y ⫽ Estimated muscle volume (cm3), using simple regression equation
Y ⫽ Estimated muscle volume (cm3), using multiple regression equation

Bandholm et al: Muscle Volume Estimates 265

Figure 2. Relationship between estimated (ultrasound and anthropometry) and measured (water displacement) muscle volume, using simple (A and B)
and multiple (C and D) regression analyses for the gastrocnemius and soleus muscles, respectively. Dotted line represents line of identity. MV, muscle
volume; SEE, standard error of the estimate.
respectively, of the variation in gastrocnemius muscle
volume, and 28% and 38%, respectively, of the variation
in soleus muscle volume (P ⬍ 0.05). The muscle-volume
prediction equations, based on multiple regression analy-
ses, are given in Table 1. Using these equations to estimate
muscle volume resulted in an r2 and SEE of 0.497 (P ⫽
0.016) and 21.9 cm3 (10.5%) for the gastrocnemius
muscle, and an r2 and SEE of 0.650 (P ⫽ 0.003) and 28.4
cm3 (10.8%) for the soleus muscle when validated against
the measured muscle volume (Fig 2C,D). No systematic
differences existed between the estimated and measured
muscle volume (P ⬎ 0.05), and the Bland and Altman
plots showed no significant correlation between muscle-
volume differences and the means for any of the two
muscles (r ⬍ 0.246, P ⬎ 0.465, Fig 3C,D).
Discussion
The main finding of the present study was that gastroc-
nemius and soleus muscle volume could be estimated with
266 PEDIATRIC NEUROLOGY Vol. 37 No. 4
standard errors of estimates between 10-13% of the
measured mean muscle volume, using ultrasonography
and anthropometry. The established muscle-volume pre-
diction equations were based on simple and multiple
regression analyses, using muscle thickness and lower leg
length as independent variables.
The accuracy of the established muscle-volume predic-
tion equations in predicting the measured muscle volume
differed somewhat between equations based on simple and
multiple regression analyses. Equations based on multiple
regression analyses produced higher r2 and lower SEEs
than equations based on simple regression analyses when
validated. This implies that the assumption of proportion-
ality with squared muscle thickness (simple regression
analyses) is not entirely valid for the gastrocnemius and
soleus muscles. By performing multiple regression analy-
ses, the accuracy of muscle-volume prediction equations
in predicting the measured muscle volume improved when
compared with simple regression analyses. This outcome
was also found by Miyatani et al., who established simple
Figure 3. Relationship between muscle volume differences (measured ⫺ estimated MV) and means (means of measured and estimated MV) with
corresponding limits of agreement (mean difference ⫾ 2 SD, solid and dashed lines, respectively), using simple (A and B) and multiple (C and D)
regression analyses for the gastrocnemius and soleus muscles, respectively. MV, muscle volume.

and multiple regression equations based on ultrasound
measurements to estimate the muscle volume of the whole
plantar flexor muscle group determined from multiple
magnetic resonance imaging scans [14]. Using only mus-
cle thickness in the simple regression equation resulted in
a SEE of 9.2%, whereas using muscle thickness and lower
leg length in the multiple regression equation resulted in a
SEE of 6.6% in a validation sample of 14 healthy men
[14]. A likely reason for this between-study difference in
results for the SEE of muscle-volume estimates might be
that the shape of the individual plantar flexor muscle
varies to a greater extent between individuals than that of
the entire plantar flexor muscle group. Furthermore, a few
precautions must be taken when interpreting the present
results.
First, the selection of investigated legs was based on
availability. No information about cause of death, age, sex,
and physical-activity status of the deceased could be
obtained. Secondly, extrapolating results obtained in ca-
davers to living humans should always be undertaken with
caution. Freezing of human tissue seems to increase the
extracellular area due to cell shrinkage [19]. This increase
is likely caused by a loss of water from the cells to the
surrounding medium (extracellular media). Hence, abnor-
mal fluid shifts within the investigated muscles might have
taken place because of the freeze-thaw cycle. It is un-
known, however, if these shifts had any impact on muscle-
thickness measurements by changing the total muscle
water content. If so, this was likely a systematic error.
Bearing this issue in mind, using the muscle-volume
prediction equations developed in the present study to
predict gastrocnemius and soleus muscle volume in living
humans may influence the SEE. The calculated SEE is
valid only for the sample in this validation study, as the
variation in the prediction equations likely varies from
sample (cadaver) to sample (living human).
Despite these apparent limitations, SEEs of 10.5% and
10.8% for the respective gastrocnemius and soleus muscle

Bandholm et al: Muscle Volume Estimates 267

volume estimates are comparable to those found using
ultrasound measurements and anthropometry to estimate
knee extensor muscle volume in living humans (SEE ⫽
11.9% [16]). With SEEs of muscle-volume estimates
around 11%, as observed in the present study, the ability to
detect intrasubject changes in muscle volume due to
muscle hypertrophy or atrophy seems limited. However, it
was recently demonstrated that 12 months of physical
training induced a 22% increase in lateral gastrocnemius
muscle volume in elderly males [20]. Hence, training-
induced, long-term hypertrophy might be detected in some
cases, based on the present data.
The Bland and Altman plots [18] revealed that the
differences between estimated and measured muscle vol-
ume were not related to the muscle volume. Hence, it
seems that the accuracy of the models in predicting the
measured muscle volume is not related to the size of the
two muscles. This finding makes it likely that the models
are also valid when applied to smaller muscle volumes
than those investigated in the present study, as would
likely be the case for children with cerebral palsy. It is
possible that gastrocnemius and soleus muscle-volume
estimates may have implications for improving dosing
standards of BTA when injected in the gastrocnemius and
soleus muscles of these patients.
Theoretically, the postinjection intramuscular distribu-
tion of BTA and the subsequent chemical denervation of
the muscle might be related to the volume of the injected
muscle, even though the motor endplate band is targeted
specifically [21,22]. Some relationship between overall
size of the muscle and regional distribution of BTA at the
injection site is assumed in the current treatment recom-
mendations, in so far as the BTA dosage for a muscle is
calculated from the total body weight of the patient.
Therefore, even though BTA is injected into the endplate
band region of a muscle, the subsequent antispastic effect
may be better dose-related to the total muscle volume than
to total body weight. This possibility should be investi-
gated in future studies, to establish an optimal pediatric
BTA dose which would reduce the cost of treatment and
possibly reduce the development of immune responses.
Hence gastrocnemius and soleus muscle-volume predic-
tion equations should be developed using magnetic reso-
nance imaging (muscle-volume criterion) and ultrasound
in a large sample of children (preferably with cerebral
palsy). Knowledge of the variation in gastrocnemius and
soleus muscle volume in children with cerebral palsy could
provide a better basis for determining the clinically relevant
accuracy of muscle-volume estimates of these muscles.
In conclusion, the present data indicate that it is possible
to estimate the volume of individual plantar flexor muscles
(gastrocnemius and soleus muscles) using ultrasound-
based measurements of muscle thickness and a measure-
ment of lower leg length in cadaveric human legs. The
standard error of these muscle-volume estimates was
between 10-13% of the measured mean muscle volume.
Based on these results, future studies could investigate if
268 PEDIATRIC NEUROLOGY Vol. 37 No. 4
similar muscle-volume prediction equations can be estab-
lished in children with cerebral palsy. For this purpose,
magnetic resonance imaging could be used as the muscle-
volume criterion.
The authors thank Derek Curtis, RPT, for comments on the manuscript.
The project was supported by grants from the Ludvig og Sara Elsass
Foundation (Charlottenlund, Denmark), the Lundbeck Foundation
(Hellerup, Denmark), the Research Foundation for Copenhagen Univer-
sity Hospital (Copenhagen, Denmark), the Danish Foundation for Re-
search in Physiotherapy (Copenhagen, Denmark), and the Britta Holles
Foundation (Copenhagen, Denmark).
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