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Original article 1
0342-5282 Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved. DOI: 10.1097/MRR.0000000000000390
Reliability of muscle thickness measurements in
ultrasonography
Nikolaos Barotsisa, Panagiotis Tsiganosb, Zinon Kokkalisc,
George Panayiotakisd and Elias Panagiotopoulosa,c
This study aims to clarify some of the issues associated
with the reliable measurement of muscle thickness on
ultrasonographic images of the musculoskeletal system,
namely the repeatability of measurements in different
time frames, the effect of body side selection, and
the effect of scan orientation. Ultrasound scans were
performed on muscles associated with essential daily
activities: geniohyoid, masseter, anterior arm muscles,
rectus femoris, vastus intermedius, tibialis anterior, and
gastrocnemius. Measurements of the muscle thickness
were performed and repeated after 1, 6, and 24 h, on both
dominant and nondominant side, using both transverse
and longitudinal scans. Thirteen healthy volunteers (eight
males and five females, mean age = 24 years, SD = 2.86,
range = 19–29) were included. The intraclass correlation
coefficient (ICC) was calculated between the baseline
and the 1-, 6-, and 24-h interval, using a two-way mixed
model of absolute agreement. The ICC ranged from 0.295
for the longitudinal scan of the left masseter muscle in
the 6-h interval to 0.991 for the longitudinal scan of the
nondominant anterior arm muscles in the 24-h interval.
The results indicate that there is variable reliability of the
measurements depending on the muscle, time frame,
body side, and scan orientation. Consequently, the
choice of these parameters can affect the validity of the
measurements. Further investigation on a larger scale is
required to establish the preferred parameters for each
anatomical site. International Journal of Rehabilitation
Research XXX: 000–000 Copyright © 2020 Wolters Kluwer
Health, Inc. All rights reserved.
International Journal of Rehabilitation Research 2020, XXX:000–000
Keywords: geniohyoid, lower limb, masseter, muscle, reliability, sarcopenia,
thickness, ultrasonography, upper limb
aRehabilitation Department, University Hospital of Patras; bClinical Radiology
Laboratory; cOrthopaedic Department and dDepartment of Medical Physics,
School of Medicine, University of Patras, Patras, Greece
Correspondence to Nikolaos Barotsis, MD, Rehabilitation Department, University
Hospital of Patras, 26504 Patras, Greece
Tel: +306973032802; fax: +302285500000; e-mail: nbarotsis@upatras.gr
Received 28 October 2019 Accepted 2 December 2019
Introduction
Quantication of skeletal muscle size changes might
be used to evaluate muscle function and document the
effectiveness of rehabilitation interventions designed to
limit muscle atrophy (English et al., 2012). The quantita-
tive measurement of skeletal muscle mass is mandatory
for the diagnosis of disorders such as sarcopenia in older
individuals (Cruz-Jentoft et al., 2019). It has been sug-
gested that ultrasound muscle thickness measurement
may be a useful tool for the early detection and monitor-
ing of sarcopenia (Can et al., 2017).
Ultrasound is a low-cost, fast, noninvasive, and widely
available imaging modality, which does not expose the
subject to ionizing radiation (Sergi et al., 2016). Apart
from extremity muscles, the diaphragm and head muscles
can also be easily assessed by ultrasound (Özçakar et al.,
2018). Moreover, ultrasound is considered to be superior
to other imaging modalities for a number of pathologies
of the musculoskeletal system due to higher resolution
and features such as dynamic imaging and simultaneous
comparability (Foti et al., 2013). The usefulness of quan-
titative ultrasound measurements for the estimation of
muscle mass loss and the detection of structural abnor-
malities is still under investigation. Commonly used
measurements include muscle thickness, cross-sectional
area, fascicle length, pennation angle, and echo-inten-
sity (Ticinesi et al., 2017). Measuring muscle thickness
can provide an estimate of the reduction in lean body
mass (Pillen and van Alfen, 2011). Thickness and fascicle
length values of medial gastrocnemius muscle have been
proposed as alternative measurements for diagnosing/
quantifying sarcopenia (Takai et al., 2014).
A drawback of the ultrasonographic measurement tech-
niques is that they present a degree of examiner depend-
ency (Tosato et al., 2017). It has also been noted that
changes in the ultrasound transducer orientation may
result in measurement errors when estimating muscle
size and ultrastructure features, such as the pennation
angle (Harris-Love et al., 2014).
There is a moderate amount of low-level evidence that
real-time brightness-mode ultrasound presents good reli-
ability for measuring muscle size across a number of limb
sites in healthy populations of children and adults, while
limited evidence is reported for the reliability of ultra-
sound measures of muscle size in clinical populations
(English et al., 2012). A recent systematic review shows
that ultrasound is a reliable and valid tool for the assess-
ment of muscle size in older adults (Nijholt et al., 2017).
Copyright © 2020 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
2 International Journal of Rehabilitation Research 2020, Vol XXX No XXX
However, to the best of our knowledge, studies on quan-
titative ultrasound muscle measurements have not taken
into consideration variables that uctuate throughout the
day and could potentially inuence muscle thickness
such as the hydration status and physical activity preced-
ing ultrasound examination. Moreover, the majority of
published studies have either used transverse or longitu-
dinal muscle scans without scientic evidence to justify
their choice. Another issue that still remains unclear is
the comparative reliability of muscle thickness measure-
ments between dominant and nondominant sides.
The aim of this study was three-fold: rst, to evaluate the
reliability of repetitive muscle thickness measurements
on ultrasound scans acquired, at different times of the
day (morning and afternoon) and at the same time of the
day, within 2 consecutive days; second, to compare the
reliability of repetitive muscle thickness measurements
between dominant and nondominant sides; and third,
to assess the reliability of ultrasound muscle thickness
measurements performed on transverse vs. longitudinal
scans.
Methods
Subjects
Young healthy individuals were recruited through an
announcement at the University of Patras Campus.
Exclusion criteria were as follows: participation in com-
petitive sports on a professional level, history of limb
fracture or surgery during the last 12 months, neuromus-
cular, metabolic, endocrine, autoimmune, cardiovascular,
respiratory, renal and hepatic diseases, and medication,
which could potentially affect muscle architecture and
echogenicity (steroids, nonsteroidal anti-inammatory
drugs, diuretics, anabolic drugs, and other hormones).
The study was approved by the Ethics Committee of the
University Hospital of Patras and was conducted accord-
ing to the Declaration of Helsinki. All participants were
informed in detail regarding the study and their formal
consent was obtained.
Ultrasound examination
Ultrasound images were acquired using the GE Logiq
P9 ultrasound system equipped the ML6-15 linear array
transducer (GE Healthcare GmbH, Freiburg, Germany).
To avoid alteration of image characteristics by software
processing, all image optimization modes were switched
off, except the tissue harmonic imaging. The gain was set
to 50, the dynamic range at 66 dB, the frequency at 10
MHz and all of them were kept constant throughout the
examination. The depth was set at 4 cm for all muscles
except for the rectus femoris and vastus intermedius,
which was set at 6 cm and facial muscles, which were set
at a depth of 3 cm. Whenever required, the depth was
increased so as to include the whole muscle in the image.
The focal zones (up to six) were distributed evenly along
the depth of the image.
A generous quantity of ultrasound gel (CLEAR ECO
Supergel, Ceracarta S.p.A., Italy) was used to achieve the
optimal ultrasound beam coupling and prevent deforma-
tion of the soft tissues due to transducer pressure during
examination. The ultrasound examination of all subjects
was performed by the same experienced musculoskeletal
sonographer (N.B.). To obtain the images in a standard
and uniform manner, the transducer was placed perpen-
dicular to the skin and eventually slightly angled (in the
elevational direction) so as to achieve the brightest echo
from muscle fascia.
As the goal of this study was to assess muscles involved
in standing, ambulation, upper limb function, and swal-
lowing, the scans were performed on the following ana-
tomical sites: for the anterior arm muscles at two-thirds
of the distance from the acromion to the elbow crease;
for the rectus femoris muscle halfway along the line from
the anterior–superior iliac spine to the superior pole of
the patella; for the tibialis anterior muscle at one-quar-
ter of the distance from the inferior pole of the patella
to the malleolus lateralis and at the bulkiest part of the
medial head of gastrocnemius muscle. The volunteers
were examined lying in prone position for the gastroc-
nemius scans with the foot hanging off the examination
bed and in a supine position for all other muscle groups
of the lower and upper limbs. They were instructed to
remain completely relaxed during ultrasound scanning
and image recording, with the upper and lower limbs
extended. Masseter muscle was examined at rest, with
the subject in sitting position. The probe was placed par-
allel to the mandibular margin, perpendicular to the skin
surface, approximately midway between the zygomatic
arch and the mandibular angle. The thickness of the
masseter was measured at its thickest part in both trans-
verse and longitudinal planes. Transverse and longitudi-
nal sonograms were recorded from all muscles bilaterally.
First, the transverse ultrasound section was acquired,
and then the transducer was rotated to 90º to acquire the
longitudinal section. The geniohyoid muscle thickness
was measured with the transducer placed on the sagit-
tal plane, between the symphysis menti and hyoid bone,
with the subject positioned as for the examination of the
masseter muscles.
All measurements were repeated within 1 hour, at 6 hours
and at 24 h from the initial examination. The anatomical
sites for all measurements were redened at each exam-
ination. Between the rst two examinations, the subjects
were instructed to remain seated and completely relaxed,
without performing any physical activity or consumption
of food and drinks as these two measurements were used
for the assessment of intrarater reliability. The subjects
were allowed to perform their usual daily activities with-
out any restriction for the examinations performed at 6 h
and at 24 h, so as to investigate the inuence of daily liv-
ing activities, hydration status, sleep, and other unfore-
seen factors on muscle thickness measurements.
Copyright © 2020 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
Muscle thickness measurements Barotsis et al. 3
Measurement of muscle thickness
Muscle thickness was measured with the built-in elec-
tronic calipers of the scanner (Figs.1 and 2). The gen-
iohyoid muscle was measured between the deep and
supercial muscle fascia; the masseter muscle between
the cortex of the mandible and the supercial fascia of
the masseter. The anterior arm muscles were measured
between the cortex of the humerus and the supercial
fascia of the biceps. It must be noted that this measure-
ment included the biceps brachii and the underlying bra-
chialis muscle. The thickness of the rectus femoris was
measured between its deep and supercial fascia; the
vastus intermedius between the cortex of the femur and
the supercial fascia of the vastus intermedius; the tibia-
lis anterior between the interosseous membrane (next to
the tibia) and the supercial fascia of the tibialis anterior;
and the medial head of gastrocnemius between its deep
and supercial fascia.
Analysis
The test–retest reliability for repeated measurements
of ultrasound muscle thickness within 1 h, at 6 and 24 h
from the initial examination was analyzed. The intraclass
correlation coefcient (ICC) was calculated, using a two-
way mixed model of absolute agreement. The ICC can
range from 0.00 (no stability/agreement) to 1.00 (perfect
agreement). An ICC of 0.70 is considered to be accept-
able (Munro, 2000).
Results
Thirteen healthy volunteers (eight males and ve
females) with a mean age of 24 years (SD = 2.86, range
= 19–29) were included in our study. Table 1 presents
the ICC for the repeated measurements of ultrasound
muscle thickness within 1 h, at 6 and 24 h from the initial
examination, with condence intervals (in parenthesis).
Discussion
To the best of our knowledge, this is the rst study that
investigates the reliability of muscle thickness measure-
ments of head and limb muscles, involved in swallowing,
activities of daily living and ambulation, with repeated
measurements at intervals of 1, 6 and 24 h on transverse
and longitudinal ultrasound sections from both the dom-
inant and nondominant side of the body.
Head muscles
The results of our study, concerning head muscles,
showed excellent reliability of geniohyoid thickness
measurements at 1, 6 and 24 h. Shimizu et al. (2016)
reported very good intratester and retest reliabilities of
geniohyoid thickness measurements of healthy volun-
teers at 1 week, which is in accordance to our results.
Contrariwise, we found that repeated thickness measure-
ments of the right masseter muscle at 1 hour, on both
transverse and longitudinal sections, and measurements
of the left masseter on transverse section, presented poor
Fig. 1
Thickness measurement of the head and upper limb muscles. The images present the placement of the electronic calipers for the geniohyoid mus-
cle (a); masseter in the transverse (b) and longitudinal section (c); anterior arm muscles in the transverse (d) and longitudinal section (e).
Copyright © 2020 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
4 International Journal of Rehabilitation Research 2020, Vol XXX No XXX
reliability. The left masseter muscle on the longitudi-
nal section showed good reliability at 1 hour but poor to
moderate in all other examinations. The overall low reli-
ability of the masseter muscle thickness measurements
could be explained by the sampling technique used in
our study, which was based on subjective transducer
positioning rather than using precise anatomical land-
marks. Moreover, it has to be taken into consideration
that the images were acquired with the jaw in relaxed
position. According to a recent review on masseter mus-
cle measurement performed by ultrasound, the lower
reproducibility of measurements in the relaxed position
in comparison with contraction could be explained by the
susceptibility of the masseter to pressure caused by the
transducer (Reis Durão et al., 2017).
Muscles of the upper limb
Thickness measurement of the anterior arm muscles
acquired from dominant and nondominant sides, on both
transverse and longitudinal sections presented excellent
reliability at 1, 6 and 24 h. These results suggest that the
reliability of thickness measurements does not depend
either on the side or the type (longitudinal or trans-
verse) of ultrasound section. Therefore, future quantita-
tive ultrasound studies could rely on one section of the
anterior arm muscles only (longitudinal or transverse)
from either dominant or nondominant side, to reduce
examiners’ workload and examination time. Moreover, it
seems that factors such as physical activity, sleep, hydra-
tion status do not affect muscle thickness on short term.
In accordance with our results, Ishida et al. (1992) have
reported that B-mode ultrasound is a highly reliable
method for the measurement of biceps brachii muscle
thickness in young adults. Their study was conducted
on transverse ultrasound section of one upper limb only,
with the examinee in standing position. Their measure-
ments were performed on two different days without giv-
ing details on the exact timing of image acquisition.
Muscles of the lower limb
Our study revealed an excellent reliability for all repet-
itive rectus femoris thickness measurements obtained
from transverse sections on both the dominant and
nondominant sides. On the contrary, the reliability of
Fig. 2
Thickness measurement of lower limb muscles. The images present the placement of the electronic calipers for the medial head of gastrocnemius
in the transverse (a) and longitudinal section (b); tibialis anterior in transverse (c) and longitudinal section (d); quadriceps femoris in the transverse
(e) and longitudinal section (f). RF, rectus femoris; VI, vastus intermedius.
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Muscle thickness measurements Barotsis et al. 5
thickness measurement was found lower on the longi-
tudinal sections, which is in accordance with the study
published by Santos and Armada-da-Silva (2017), who
concluded that quadriceps muscle thickness measured
on longitudinal scans are somewhat less reliable com-
pared to transverse scans. However, the difference in the
reliability between the transverse and longitudinal scans
in our study was not statistically signicant (P values for
repetitive measurements at 1, 6 and 24 h ranged from
0.107 to 0.228 for the dominant side, and from 0.343 to
0.761 for the nondominant side).
The reliability of vastus intermedius thickness measure-
ment on transverse and longitudinal sections, acquired
from both dominant and nondominant sides, was good
at 1 hour from the initial measurement, but all other
measurements at 6 and 24 h presented lower reliability.
Tillquist et al. (2014) measured the thickness of quadri-
ceps muscle in healthy volunteers and reported excel-
lent intra- and inter-rater reliability. Their examination
protocol was based on the average thickness of left and
right quadriceps, using two readings for each side (i.e. at
two different anatomical locations), which was performed
exclusively on transverse sections. The authors have not
performed thickness measurements on individual heads
of the quadriceps muscle. Agyapong-Badu et al. (2014)
have measured quadriceps thickness, with two measure-
ments at 1 week apart, using transverse sections of the
anterior thigh and reported excellent reliability. Strasser
et al. (2013) has demonstrated that measurements of
muscle thickness were highly reproducible in all heads
of quadriceps muscle in young individuals, using two
measurements each day for two separate days. However,
the time of the day the examinations took place and the
interval of time between repeated measurements are not
reported and thickness of quadriceps heads was measured
on transverse images only. It should be noted that the
data collection protocol followed in our study differs from
the one used by Strasser, which could partially explain
the discrepancy in our results concerning vastus interme-
dius thickness. Our measurements were performed with
the patient lying in the supine position with the knee
fully extended, and the quadriceps muscle relaxed. We
assume that this positioning does not effectively restrict
thigh rotation, which could probably explain the varia-
bility of the vastus intermedius thickness measurements.
On the contrary, if the knee is slightly exed (supported
by a roll or pillow), thigh rotation is limited, which most
probably could allow for more precise repetitive thick-
ness measurements. Additionally, the irregular shape of
vastus intermedius muscle in transverse section, in con-
trast to the regular elliptical shape of the rectus femoris,
makes the accurate placement of calipers inconsistent in
repetitive measurements. Moreover, conditions such as
hydration status, physical activity and rest, which vary
between repetitive measurements, could have contrib-
uted to the decrease in the muscle thickness measure-
ment reliability.
In our study, repeated thickness measurements of tibialis
anterior at 1, 6, and 24 h, acquired from both longitudinal
and transverse sections presented excellent reliability for
the nondominant side. The reliability on the dominant
side was found to be excellent for all longitudinal sections.
Table 1 Intraclass correlation coefficient for the repeated measures of ultrasound muscle thickness measurements with confidence
intervals (in parenthesis)
0–1 h 0–6 h 0–24 h
GHY 0.943 (0.824–0.982)d0.936 (0.774–0.983)d0.942 (0.786–0.985)d
RT MAS LONG 0.411 (−0.195 to 0.779)a0.736 (0.216–0.928)b0.694 (0.133–0.915)b
RT MAS TR 0.522 (0.035–0.820)d0.748 (0.257–0.931)b0.574 (−0.088 to 0.877)b
LT MAS LONG 0.771 (0.406–0.924)c0.295 (−0.265 to 0.746)a0.660 (0.132–0.901)b
LT MAS TR 0.358 (−0.260 to 0.754)a0.489 (−0.216 to 0.847)a0.738 (0.266–0.927)b
D AAM LONG 0.976 (0.926–0.993)d0.936 (0.778–0.984)d0.980 (0.923–0.995)d
D AAM TR 0.966 (0.895–0.989)d0.978 (0.915–0.994)d0.966 (0.876–0.991)d
ND AAM LONG 0.979 (0.933–0.993)d0.987 (0.947–0.997)d0.991 (0.966–0.998)d
ND AAM TR 0.975 (0.919–0.992)d0.970 (0.892–0.992)d0.963 (0.857–0.991)d
D RF LONG 0.852 (0.587–0.952)c0.950 (0.819–0.987)d0.944 (0.750–0.987)d
D RF TR 0.960 (0.798–0.989)d0.964 (0.867–0.991)d0.976 (0.902–0.994)d
ND RF LONG 0.855 (0.604–0.953)c0.621 (0.010–0.891)b0.871 (0.587–0.966)c
ND RF TR 0.909 (0.727–0.971)d0.960 (0.854–0.990)d0.971 (0.890–0.993)d
D VI LONG 0.895 (0.693–0.967)c0.570 (−0.056–0.873)b0.411 (−0.322 to 0.818)a
D VI TR 0.896 (0.704–0.967)c0.583 (0.009–0.875)b0.464 (−0.252 to 0.838)a
ND VI LONG 0.880 (0.653–0.962)c0.936 (0.770–0.984)d0.805 (0.399–0.948)c
ND VI TR 0.867 (0.622–0.958)c0.938 (0.785–0.984)d0.839 (0.475–0.957)c
D TA LONG 0.915 (0.750–0.973)d0.976 (0.908–0.994)d0.955 (0.829–0.989)d
D TA TR 0.955 (0.864–0.986)d0.953 (0.824–0.988)d0.875 (0.569–0.968)c
ND TA LONG 0.919 (0.766–0.974)d0.984 (0.938–0.996)d0.949 (0.808–0.987)d
ND TA TR 0.976 (0.928–0.993)d0.936 (0.770–0.984)d0.936 (0.776–0.984)d
D MHG LONG 0.878 (0.658–0.961)c0.849 (0.528–0.959)c0.685 (0.113–0.912)b
D MHG TR 0.823 (0.510–0.943)c0.785 (0.372–0.941)c0.736 (0.221–0.928)b
ND MHG LONG 0.944 (0.832–0.982)d0.721 (0.237–0.921)c0.758 (0.321–0.933)c
ND MHG TR 0.903 (0.713–0.969)d0.898 (0.632–0.974)c0.886 (0.317–0.975)c
AAM, anterior arm muscles; D, dominant side; GHY, geniohyoid; ICC, intraclass correlation coefficient; LONG, longitudinal ultrasound scan; LT, left; MAS, masseter;
MHG, medial head of gastrocnemius; ND, nondominant side; RF, rectus femoris; RT, right; TA, tibialis anterior; TR, transverse ultrasound scan; VI, vastus intermedius.
abelow 0.50: poor ICC; bbetween 0.50 and 0.75: moderate ICC; cbetween 0.75 and 0.90: good ICC dAbove 0.90: excellent ICC;.
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6 International Journal of Rehabilitation Research 2020, Vol XXX No XXX
For the transverse sections, the reliability was excellent
at 1 and 6 h but lower at 24 h. However, concerning the
transverse sections acquired in 24 h, the difference in the
reliability between the dominant and nondominant side
was not statistically signicant (P = 0.403).
The measurements acquired from the transverse sec-
tions of the medial head of gastrocnemius, from both the
dominant and nondominant sides, and the longitudinal
sections, from the nondominant side, presented excellent
reliability at 1 h, but lower degrees of reliability in sub-
sequent measurements at 6 and 24 h. This result could
be explained by changes in muscle thickness induced by
factors such as physical activity, rest, hydration/dehydra-
tion and other metabolic or endocrine factors. Therefore,
the time of the day the images have been acquired must
be clearly documented in the methodology of ultra-
sound quantitative studies and ideally kept the same
for all individuals, since it might affect the reliability of
the measurements. It is worth noticing that we acquired
the images from the bulkiest region of the muscle belly,
which could have been a reason for the overall lower reli-
ability of repetitive thickness measurement in our study.
It is important to underline the need for reliability stud-
ies using an acquisition technique based on standardized
transducer positioning so as to overcome the subjectiv-
ity of the examiner on selecting the bulkiest part of the
muscle. Moreover, it seems that body positioning during
image acquisition can inuence the reliability of thick-
ness measurements. According to Thoirs and English
(2009), measurements taken in the recumbent position
presented lower reliability compared to those recorded in
standing position.
Our study presents several limitations. The sample size is
relatively small for extracting safe conclusions, especially
as it concerns measurements with increased reliability
range. Additionally, the sample included young healthy
adults only, which does not allow the generalization of
our results to other age groups and disease states. Further
research studies are required to address these limitations
and to establish the signicance of the ndings of the
current study.
Conclusion
Despite the relatively large amount of data published
on the quantication of muscle size for the diagnosis
of sarcopenia and other muscle disorders, the reliability
of image acquisition techniques is still not adequately
investigated. Moreover, the selection of the ultrasound
scan and sampling side used in published studies does
not seem to be evidence based. The results of our study
suggest that ultrasound thickness measurements of head
and limb muscles present various degrees of reliabil-
ity, which depend on the type of section and sampling
side. Large scale studies are required to establish the
optimal examination protocols for quantitative musculo-
skeletal ultrasonography and evidence-based guidelines
are needed for the standardization of ultrasound image
acquisition and measurement techniques.
Acknowledgements
Conflicts of interest
There are no conicts of interest.
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