Reliability and validity of ultrasound measurements of

muscle fascicle length and pennation in humans: a

systematic review
Li Khim Kwah, Rafael Z. Pinto, Joanna Diong and Robert D. Herbert
J Appl Physiol 114:761-769, 2013. First published 10 January 2013;
doi:10.1152/japplphysiol.01430.2011

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Reliability and validity of ultrasound measurements of muscle fascicle length
and pennation in humans: a systematic review
Li Khim Kwah,1 Rafael Z. Pinto,2 Joanna Diong,2 and Robert D. Herbert3
1
Australian Catholic University, Sydney, New South Wales, Australia; 2The George Institute for Global Health, Sydney, New
South Wales, Australia; and 3Neuroscience Research Australia, Sydney, New South Wales, Australia
Submitted 30 November 2011; accepted in final form 9 January 2013
Kwah LK, Pinto RZ, Diong J, Herbert RD. Reliability and validity of
ultrasound measurements of muscle fascicle length and pennation in humans:
a systematic review. J Appl Physiol 114: 761769, 2013. First published
January 10, 2013; doi:10.1152/japplphysiol.01430.2011.Ultrasound im-
aging is widely used to measure architectural features of human
skeletal muscles in vivo. We systematically reviewed studies of the
reliability and validity of two-dimensional ultrasound measurement of
muscle fascicle lengths or pennation angles in human skeletal mus-
cles. A comprehensive search was conducted in June 2011. Thirty-six
reliability studies and six validity studies met the inclusion criteria.
Data from these studies indicate that ultrasound measurements of
muscle fascicle lengths are reliable across a broad range of experi-
mental conditions [intraclass correlation coefficient (ICC) and r val-
ues were always 0.6, and coefficient of variation values were
always 10%]. The reliability of measurements of pennation angles
is broadly similar (ICC and r values were always 0.5 and coefficient
of variation values were always 14%). Data on validity are less
extensive and probably less robust, but suggest that measurement of
fascicle lengths and pennation angles are accurate (ICC 0.7) under
certain conditions, such as when large limb muscles are imaged in a
relaxed state and the limb or joint remains stationary. Future studies
on validity should consider ways to test for the validity of two-
dimensional ultrasound imaging in contracted or moving muscles and
the best method of probe alignment.
ultrasonography; muscle architecture; pennation
ULTRASOUND IMAGING AND MAGNETIC resonance imaging are
widely used to measure muscle fascicle lengths and pennation
in human skeletal muscles in vivo (39, 51). Measurements of
muscle fascicle lengths and pennation are often used in phys-
iological and biomechanical modeling studies to estimate the
amount of force-generating capacity of muscles (38).
Magnetic resonance imaging has the advantages of having
high spatial resolution (39) and the ability to measure large
areas of muscles simultaneously (19, 39, 58). This is especially
useful considering the heterogeneity of muscle fascicle lengths
in at least some muscles (58). However, magnetic resonance
imaging is expensive and time-consuming (51). In all but a few
magnetic resonance imaging machines, the scanning must be
done statically with the subject in a supine, prone, or side-lying
position, limiting the dynamic measurement of muscle fascicle
lengths and pennation in other body positions or activities.
Ultrasound is less expensive, quicker, and easier to operate (39,
51). It enables measurement of muscle fascicle lengths during
contraction in various positions or activities, such as standing,
walking, and running (1, 37, 40). Innovations in ultrasound
imaging have also added to its attractiveness as a tool for
Address for reprint requests and other correspondence: R. D. Herbert,
Neuroscience Research Australia, PO Box 1165, Randwick, NSW 2031,
Australia (e-mail: r.herbert@neura.edu.au).
http://www.jappl.org 8750-7587/13 Copyright 2013 the American Physiological Society
J Appl Physiol 114: 761769, 2013.
First published January 10, 2013; doi:10.1152/japplphysiol.01430.2011.
measuring muscle fascicle lengths and pennation. For example,
extended field-of-view imaging (20, 64) has allowed observa-
tion of long muscle fascicles under static conditions (53), use
of flat ultrasound probes has made observation of muscle
fascicle lengths during activities such as walking and running
more feasible (1, 37), and tracking software has reduced the
burden of measuring fascicle lengths from video sequences
(37, 40, 45, 55). For these reasons, ultrasound is often preferred
to magnetic resonance imaging in measuring muscle fascicle
lengths and pennation in humans.
Several studies have investigated the reliability of ultrasound
measurement of muscle fascicle lengths and pennation. A
smaller number of studies have examined validity by compar-
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ing ultrasound measurement with direct measurement of fas-
cicle lengths and pennation in cadaveric muscle. To our knowl-
edge, these studies have not been comprehensively reviewed.
The aims of the present study are to systematically review
studies of the reliability and validity of ultrasound measure-
ment of human muscle fascicle lengths and pennation angles.
MATERIALS AND METHODS
Identification, screening, and inclusion of studies. The review
included studies of reliability or validity of two-dimensional (2D)
ultrasound measurement of muscle fascicle lengths or pennation
angles in human skeletal muscles. Only studies published in English
were reviewed. Studies were considered to be reliability studies, if
they reported concordance of repeated measures of any aspect of the
ultrasound measurement procedure. This included repeated ultrasound
imaging, or repeated extraction of fascicle lengths from images, or
repeated measurements by the same or different examiner. Studies
were considered to be validity studies if they compared ultrasound
measurement of fascicle lengths with measurement made directly
from the muscle in cadavers.
Search strategies were developed for three electronic databases
(Medline to June Week 1 2011, Embase 2011 Week 23, and Cinahl to
12 June 2011). Searches were conducted independently by two re-
viewers using the following search syntax.
Search syntax for Medline and Embase (Ovid):
1) (ultraso* or echography).mp.
2) (muscle*).mp.
3) (fascicle* or fibre* or fiber*).mp.
4) (pennation or pinnation).mp.
5) 1 and 2 and (3 or 4).
6) Limit 5 to animals.
7) 5 not 6.
8) Limit 7 to English language.
Search syntax in Cinahl (EBSCO):
1) ultraso*.
2) muscle*.
3) fascicle* or fibre* or fiber*.
4) pennation or pinnation.
5) S1 and S2.
6) S3 or S4.
761
762 Ultrasound Measurement of Human Muscle Fascicles
7) S5 and S6.
8) Limit 7 to English language.
Titles and abstracts of studies obtained from the search strategies
were screened by both reviewers. We also searched the reference list
of included articles and our personal libraries.
Data extraction and quality assessment. Extraction of reliability
and validity data, and assessment of study quality, were conducted
independently by two reviewers. Where there were discrepancies, a
third reviewer was consulted. The following convention (17) was used
to describe reliability or accuracy, based on correlations: little, if
any (correlations of 0.00 0.25), low (0.26 0.49), moderate
(0.50 0.69), high (0.70 0.89), and very high (0.90 1.00).
Several statistics were used, in the original study reports, to
quantify reliability and validity. We abbreviate those statistics as
follows: coefficient of multiple correlation (CMC), intraclass correla-
tion coefficient (ICC), Pearsons correlation coefficient (r), Cohens
kappa (), coefficient of variation (CV), standard error of the mea-
surement (SEM), and standard error of the measurement expressed as
a percentage of the mean (SEM%). The correlation estimates for
which the convention is used to describe reliability or validity refer to
the CMC, ICC, r, and estimates. There was rarely enough informa-
tion to allow translation of the reported statistics to a common
statistic, so we made no attempt to do so or to conduct a meta-
analysis. As data on fascicle lengths and pennation angles of human
muscles may be of intrinsic interest to physiologists, these data were
also extracted from the reliability and validity studies. We extracted
data from studies in which the muscles were relaxed and measure-
ments were obtained in anatomical joint positions (e.g. upper and
lower limbs extended, ankle at right angles to shank).
There are several checklists and scales for assessing the quality of
studies of reliability and validity in systematic reviews (22, 63), but
none is universally acknowledged as superior. To rate the quality of
reliability studies, we used most of the items in the scale designed by
Hebert and colleagues (22). Items pertaining to external validity (e.g.,
representativeness of participants and examiners in studies) were not
included, as the use of ultrasound is not limited to specific participants
or examiners in clinical practice. To rate the quality of validity
studies, we used items from the QUADAS scale (for diagnostic
accuracy studies) (62) that pertained to blinding and execution of
tests, and we added items on statistical methodology. The final
checklist is shown in Table 1.
Table 1. Quality assessment of reliability (N 36) and validity (N 6) studies
Criterion
1. Was the ultrasound measurement procedure described in sufficient detail
to permit its replication?
2. Were participants characteristics under study stable during research?
3. Were examiners blinded to each others results or to prior results?
4. Were appropriate measures used for calculating reliability?
5. Were appropriate measures used for calculating precision of estimates of
reliability?
1. Was the ultrasound measurement procedure described in sufficient detail
to permit its replication?
2. Was the anatomical inspection procedure described in sufficient detail to
permit its replication?
3. Were the measurements blinded?
4. Were appropriate measures used for calculating validity?
5. Were appropriate measures used for calculating precision of estimates of
validity?
Values are no. of studies, with percentages in parentheses.
J Appl Physiol doi:10.1152/japplphysiol.01430.2011 www.jappl.org
Kwah LK et al.
RESULTS
Identification of eligible studies. The search yielded 1,878
articles (Fig. 1). Full-text copies of 48 articles were screened.
Out of these, 10 were excluded because they did not measure
muscle fascicle lengths or pennation angles (16, 32, 40, 65),
did not report concordance of repeated measures of the ultra-
sound procedure (9, 15, 45, 55), or used three-dimensional
(3D) ultrasound (25, 33). Consequently, 38 studies satisfied the
inclusion criteria and were included in the review. Four studies
examined both reliability and validity of ultrasound in measur-
ing muscle fascicle lengths.
Data extraction. Data were extracted from 36 reliability
studies (1, 2, 6 8, 10 14, 18, 21, 23, 24, 26 28, 30, 31, 34, 36,
41 44, 46 50, 5254, 56, 57, 60) and 6 validity studies (4, 12,
28 30, 52). Characteristics of reliability and validity studies
are shown in Tables 2 and 3.
Quality assessment. For nearly all reliability studies, mea-
surement procedures were described in sufficient detail to
permit replication, the characteristics under study were judged
to have been stable across replications, and appropriate statis-
tics were used to quantify reliability. However, very few
studies stated that measurements made for one replicate were
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blinded to the other replicate, and few studies reported the
precision of estimates of reliability (Table 1).
For two-thirds of the validity studies, ultrasound measure-
ment and anatomical inspection procedures were described in
sufficient detail to permit replication. Only one of six studies
provided appropriate validity estimates. Instead, most of these
studies reported the mean measurements of the ultrasound
measures and the cadaver measures. This does not permit
evaluation of the accuracy of individual measurements. None
of the studies reported the precision of validity estimates
(Table 1).
Reliability (all). The reliability of measurements of muscle
fascicle lengths was moderate to very high (CMC 0.89
0.95, ICC 0.62 0.99, r 0.74 0.96, CV 0.0 9.8%,
Studies Satisfying
Criterion, N (%) Ref Nos.
Reliability
33 (92) 1, 2, 68, 1014, 18, 21, 24, 26, 27, 30, 31, 34, 36, 4144,
4650, 5254, 56, 60
33 (92) 1, 68, 1013, 18, 21, 23, 24, 2628, 30, 31, 34, 36,
4144, 4650, 5254, 57, 60
2 (6) 0, 43
34 (94) 1, 2, 5, 7, 8, 1014, 18, 21, 24, 2628, 30, 31, 34, 36,
4143, 4650, 5254, 56, 57, 60
5 (14) 18, 36, 47, 53, 60
Validity
4 (67) 4, 29, 30, 52
4 (67) 4, 12, 29, 52
2 (33) 28, 30
1 (17) 29
0
 Ultrasound Measurement of Human Muscle Fascicles
SEM 0 19 mm, and SEM% 4.314.2%; supplemental
data; the online version of this article contains supplemental
data). Reports of the reliability of pennation angle measure-
ments were slightly lower, but still ranged from moderate to
very high (CMC 0.84 0.90, ICC 0.511.00, r 0.76
0.95, 0.80 0.84, CV 0.0 13.5%, SEM 0.11.2, and (contracted fascicle lengths: CMC 0.89 0.95, ICC 0.62
SEM% 5.0 10.9%; supplemental data). From Supplemental
Figs. S1S6, it can be observed that the majority of studies
reported high reliability estimates (CMC, ICC, r, and 0.8,
CV 10%, SEM for fascicle length 10 mm, and SEM for
pennation angle 2). Lower reliability estimates (SEM of 17
mm and SEM% of 14.2% for muscle fascicle length, ICC of
0.51 and CV of 13.5% for pennation angle) were reported by
two studies, which measured muscle fascicle lengths and
pennation angles between ultrasound sessions performed a few
weeks apart (42, 56). One of the studies also carried out
repeated measurement of fascicle lengths and pennation angles
in muscles imaged in a contracted state (42). Another study had
also reported a low reliability estimate (SEM of 19 mm) for
muscle fascicle length; however, the ICCs for muscle fascicle
length reported in the study were considered high (ICC
0.76 0.86) (6).
Reliability (relaxed and contracted muscle states). Reliabil-
ity estimates obtained from muscles imaged in a relaxed state
were generally high to very high for fascicle lengths, and
moderate to very high for pennation angles (relaxed fascicle
lengths: ICC 0.740.99, r 0.96, CV 0.06.8%, SEM
0 19 mm, and SEM% 8.510.9%; relaxed pennation angles:
J Appl Physiol doi:10.1152/japplphysiol.01430.2011 www.jappl.org
Kwah LK et al. 763
Fig. 1. Flow diagram of literature search. 3D,
three-dimensional; US, ultrasound.
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ICC 0.621.00, r 0.87 0.95, CV 0.0 8.5%, SEM
0.11.2, and SEM% 5.0 10.2%) (2, 6 8, 14, 18, 21, 28,
36, 41 43, 46, 47, 49, 53, 54, 60). For muscles imaged in a
contracted state, reliability estimates for muscle fascicle
lengths and pennation angles were moderate to very high
0.99, CV 0.0 8.3%, SEM 117 mm, and SEM%
4.314.2%; contracted pennation angles: CMC 0.84 0.90,
ICC 0.511.00, CV 0.0 8.3%, SEM 0.9 1.2, and
SEM% 5.110.9%) (1, 11, 18, 21, 31, 34, 41, 42, 57).
Reliability (between sessions). When ultrasound measures
were repeated in different sessions, the between-session reli-
ability of muscle fascicle length measurements was moderate
to very high. Reliability of pennation angle measurements was
slightly lower, although estimates still ranged from moderate to
very high (fascicle lengths: CMC 0.93, ICC 0.62 0.99,
r 0.93 0.96, CV 2.39.8%, SEM 219 mm, and
SEM% 4.314.2%; pennation angles: CMC 0.87, ICC
0.511.00, r 0.87 0.95, CV 2.113.5%, SEM 0.21.2,
and SEM% 5.0 10.9%) (1, 2, 6, 13, 18, 28, 30, 34, 36,
41 43, 46, 48 50, 52, 53, 56, 60).
Reliability (between images). When measurements were
repeatedly obtained from the same ultrasound images, reliabil-
ity for muscle fascicle lengths and pennation angles were high
to very high (fascicle lengths: CMC 0.95, ICC 0.87 0.99,
CV 0.0 8.3%, and SEM 0 8 mm; pennation angles:
CMC 0.90, ICC 0.851.00, 0.80 0.81, CV
764 Ultrasound Measurement of Human Muscle Fascicles Kwah LK et al.

Table 2. Characteristics of reliability studies

Characteristics Details (n Studies) Ref. Nos.

Subjects Healthy (32) 1, 2, 68, 1013, 18, 21, 23, 24, 2628, 30, 31, 34, 36, 4144,
4750, 52, 53, 56, 57
Diseased/injured/received intervention (6) 2, 14, 46, 47, 54, 60
Examiners Radiologist/sonographer (3) 30, 36, 54
Not qualified/not stated examiner (33) 1, 2, 68, 1014, 18, 21, 23, 24, 2628, 31, 34, 4144, 4650,
52, 53, 56, 57, 60
Muscles examined Ssp (1), TB (1), BB (1), Br (1), EDC (1), VL (15), MG (13), Ssp (30), TB (28), BB (14), Br (24), EDC (10), VL (2, 68,
TA (4), Sol (4), LG (3), VI (2), RF (2), BF (1), VM (1) 11, 14, 21, 23, 26, 42, 47, 53, 56, 57, 60), MG (1, 13, 14,
18, 27, 34, 36, 41, 44, 46, 48, 49, 52), TA (11, 14, 31, 50),
Sol (13, 27, 44, 54), LG (27, 43, 44), VI (6, 56),
RF (6, 47), BF (12), VM (6)
State of muscle Relaxed (29) 2, 68, 10, 1214, 18, 21, 23, 2628, 30, 36, 4144, 4648,
49*, 50, 5254, 60
Contracted (19) 1*, 10, 11*, 13, 18, 21, 23, 26, 27, 30, 31*, 34*, 41, 42, 44,
48, 50, 52, 57
Method of ultrasound Stationary probe (31) 1, 2, 68, 1114, 18, 21, 23, 24, 2628, 30, 31, 34, 4144,
imaging 4749, 52, 54, 56, 57, 60
Moving probe/extended-field-of-view (5) 10, 36, 46, 50, 53
Method of fascicle length Digital (21) 1, 2, 6, 7, 1114, 18, 30, 31, 34, 36, 41, 43, 46, 49, 50, 52,
measurement 53, 60
Manual (6) 21, 24, 26, 27, 44, 54
Unclear/not stated (9) 8, 10, 23, 28, 42, 47, 48, 56, 57
Aspect of study repeated Ultrasound sessions (21) 1, 2, 6, 13, 18, 23, 28, 30, 34, 36, 4143, 46, 4850, 52, 53,
56, 60
Images (20) 1, 6, 1012, 14, 21, 24, 26, 31, 34, 36, 41, 43, 44, 47, 48, 50,

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53, 54
Raters (4) 10, 30, 53, 57
Unclear/not stated (3) 7, 8, 27
Ssp, supraspinatus; TB, triceps brachii; BB, biceps brachii; Br, brachialis; EDC, extensor digitorum communis; VL, vastus lateralis, MG, medial
gastrocnemius; TA, tibialis anterior; Sol, soleus; LG, lateral gastrocnemius; VI, vastus intermedius; BF, biceps femoris; RF, rectus femoris; VM, vastus medialis.
*Limb or joint was moving while images were obtained.

0.0 8.3%, and SEM 0.3 0.6) (1, 6, 10, 11, 14, 21, 24, 26,
31, 34, 36, 41, 43, 47, 48, 50, 53, 54).
Reliability (between raters). Fewer studies investigated re-
liability between raters. When two raters obtained measures
from the same muscles, the reliability of the measurement of
muscle fascicle lengths and pennation angles was high to very
high (fascicle lengths: ICC 0.80 0.97, r 0.74, CV
3.1%; pennation angles: ICC 0.80, r 0.76, and 0.84)
(10, 30, 53, 57).
Reliability (muscles). Vastus lateralis and medial gastrocne-
mius were the muscles most commonly imaged for reliability.
The reliability of ultrasound imaging was high to very high for
supraspinatus (fascicle lengths: r 0.74 0.93, pennation
angles: r 0.76 0.88) (30), triceps brachii (pennation angles:
r 0.87 0.95) (28), biceps brachii (pennation angles: ICC
0.99, SEM 0.2) (14), brachialis (pennation angles: ICC
0.89) (24), extensor digitorum communis (pennation angles:
0.80 0.84) (10), medial gastrocnemius (fascicle lengths:

Table 3. Characteristics of validity studies

Characteristics Details (n Studies) Ref. Nos.

Examiners for ultrasound Qualified radiologist (1) 30

Not qualified/not stated (5) 4, 12, 28, 29, 52
Muscles examined Ssp (1), TB (1), BF (2), ST (1), MG (2) Ssp (30), TB (28), BF (12, 29), ST (29), MG (4, 52)
Determination of plane of muscle fascicles during Probe placed perpendicular to skin (3) 4, 28, 52
ultrasound
Optimized image quality (1) 12
Unclear/not stated (2) 29, 30
Observation of muscle fascicle lengths from Full field-of-view imaging (5) 12, 2830, 52
ultrasound image
Extrapolation (1) 4
Determination of plane of muscle fascicles during Checked for cut fascicles (1) 4
anatomical inspection
Fibers dissected out (3) 12, 29, 52
Unclear/not stated (2) 28, 30
Determination of same muscle fascicles as Marked with pins (1) 29
observed with ultrasound
Marked on skin (1) 52
3D microscope (1) 30
Unclear/not stated (3) 4, 12, 28
ST, semitendinosus.

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 Ultrasound Measurement of Human Muscle Fascicles Kwah LK et al. 765

Table 4. Muscle fascicle lengths obtained via anatomical inspection and ultrasound procedures
Muscles Anatomical Inspection, mm Ultrasound, mm Ref. Nos.

Supraspinatus
Anterior middle 54 9* 54 3* 30
59 8
Anterior deep 58 4* 57 5*
59 9
Posterior deep 30 2* 30 2*
Anterior 59 7
Posterior 36 6
Extensor digitorum communis 73 24 (men) 10
50 9 (women)
Vastus lateralis
Entire length 79 8 53
80 8
80 9
83 7
Middle 72 22 (cerebral palsy) 47
75 12 (normal)
84 14 (pretraining, training group) 2
82 12 (pretraining, control group)
92 15 (posttraining, training group)
85 11(posttraining, control group)
57 (nonoperated) 7
65 (operated)

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51 3* (right)
50 3* (left)
Rectus femoris
Middle 71 21 (cerebral palsy) 47
98 23 (normal)
Biceps femoris
Long head 76 10 80 17 29
80 15 88 18 12
Semitendinosus 187 38 204 39 29
Soleus
Anterior, proximal, and middle 27 6 44
Posterior, middle, and distal 25 4
Middle 29 5 27
Medial gastrocnemius 36 (nonparetic leg) 46
30 (paretic leg)
Proximal 40 3* 40 2* (cadaver) 52
49 5 (live subjects)
Middle 30 4 (Phillips HD11 US machine) 36
30 4 (Chison 8300 US machine)
40* 40* 4
44* 42*
43* 48*
27* 32*
37 (nonparetic leg) 46
34 (nonparetic leg)
31 (paretic leg)
29 (paretic leg)
40 5 27
37 2* 35 2* (cadaver) 52
51 4 (live subjects)
53 8 (live subjects)
Distal 36 2* 31 4* (cadaver)
59 7 (live subjects)
Proximal and distal 34 7 44
Lateral gastrocnemius 40 (nonparetic leg) 46
33 (paretic leg)
Proximal 42 (nonparetic leg)
34 (paretic leg)
Middle 40 (nonparetic leg)
32 (paretic leg)
47 7 27
Distal 43 10 44
Values are means or means SD. *Individual raw data (n 1). Ultrasound values for presumed planes were extracted. Data from children.

J Appl Physiol doi:10.1152/japplphysiol.01430.2011 www.jappl.org

766 Ultrasound Measurement of Human Muscle Fascicles Kwah LK et al.

Table 5. Muscle pennation angles obtained via anatomical inspection and ultrasound procedures
Muscles Anatomical Inspection, Ultrasound, Ref. Nos.

Supraspinatus
Anterior middle 109 31 30
Anterior deep 142 43
Anterior 121 31
Triceps brachii
Long head 14* 14* 28
16* 15*
10* 11*
9* 10*
33 16 (body builder)
15 6 (normal)
Medial head 19 8 (body builder)
11 5 (normal)
Brachialis 92 24
Extensor digitorum communis 7 1 (men) 10
6 1 (women)
Vastus lateralis
Middle 10 3 (cerebral palsy) 47
14 3 (normal)
16 3 (pretraining, training group) 2
16 2 (pretraining, control group)
16 3 (posttraining, training group)
15 2 (posttraining, control group)

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21 (nonoperated) 7
15 (operated)
20 1* (right)
20 1* (left)
Rectus femoris
Middle 12 3 (cerebral palsy) 47
13 3 (normal)
Biceps femoris
Long head 14 2 14 3 29
15 3 (superficial) 14 3 (superficial) 12
17 4 (deep) 14 3 (deep)
Semitendinosus 13 3 13 3 29
Soleus
Anterior, proximal, and middle 46 11 (anterior angle) 44
Posterior, middle, and distal 30 7 (anterior angle)
25 5 (posterior angle)
Middle 29 3 27
Medial gastrocnemius 19 (nonparetic leg) 46
18 (paretic leg)
Proximal 16 2* (superficial, cadaver) 14 2* (superficial, cadaver) 52
21 4* (deep, cadaver) 20 2* (deep, cadaver)
16 6 (live subjects)
22 4 (live subjects)
Middle 22 3 (Phillips HD11 US machine) 36
21 3 (Chison 8300 US machine)
11* 12* 4
18* 19*
27* 26*
25* 20*
18 (nonparetic leg) 46
20 (nonparetic leg)
20 (paretic leg)
23 (paretic leg)
31 3 27
20 2* (superficial, cadaver) 19 2* (superficial, cadaver) 52
21 2* (deep, cadaver) 19 1* (deep, cadaver)
17 3 (live subjects)
21 5 (live subjects)
Distal 16 3* (superficial, cadaver) 13 3* (superficial, cadaver) 52
19 3* (deep, cadaver) 17 2* (deep, cadaver)
13 2 (live subjects)

17 2 (live subjects)
Continued

J Appl Physiol doi:10.1152/japplphysiol.01430.2011 www.jappl.org

 Ultrasound Measurement of Human Muscle Fascicles
Table 5.Continued
Muscles Anatomical Inspection,
Proximal and distal 33 5 (anterior angle)
32 6 (posterior angle)
Lateral gastrocnemius
Middle
Distal 21 3 (anterior angle)
17 3 (posterior angle)
Values are means or means SD. *Individual raw data (n 1). Ultrasound values for presumed planes were extracted. Data from children.
CMC 0.93 0.95, ICC 0.81 0.99, r 0.96, CV
0.0 9.8%, and SEM 0 mm; pennation angles: CMC
0.87 0.90, ICC 0.851.00, CV 0.0 9.8%, and SEM
0.2) (1, 14, 18, 34, 36, 41, 46, 48, 49, 52), tibialis anterior
(fascicle lengths: ICC 0.93 0.99, CV 2.8 8.3%, and
SEM 1 mm; pennation angles: ICC 0.99, CV 8.3%, and
SEM 0.1) (11, 14, 31, 50), and rectus femoris (fascicle
lengths: ICC 0.96 0.98; pennation angles: ICC 0.95
0.97) (47). Reliability was moderate to very high for vastus
lateralis (fascicle lengths: ICC 0.62 0.99, CV 0.0 6.8%,
SEM 0 17 mm, and SEM% 4.314.2%; pennation
angles: ICC 0.511.00, CV 0.0 7.5%, SEM 0.21.2,
and SEM% 5.0 10.9%) (2, 7, 8, 11, 14, 21, 26, 42, 47, 53,
57, 60). There were not enough data to conclude on soleus
(pennation angles: SEM 0.5) (54) and lateral gastrocnemius
(pennation angles: CV 4.2 8.5%) (43). Studies that mea-
sured reliability of ultrasound imaging for vastus intermedius
(6, 56), biceps femoris (12), and vastus medialis (6) did not
report reliability estimates specific to the muscles.
Validity. Only one of six validity studies provided appropri-
ate validity estimates (29). The remaining studies compared
means of measurements obtained during ultrasound imaging
procedures with means obtained during anatomical inspection
procedures (4, 12, 28, 30, 52). This provides a measure of
mean bias, but not of the accuracy of individual measures. As
two studies provided individual cadaveric data of fascicle
lengths and pennation angles, we calculated ICC estimates
from their data (4, 28). The data from these two small studies
(N 4 and N 3) and one additional study (N 3), which
reported ICC estimates, indicated that the accuracy of mea-
surements of fascicle lengths and pennation angles was high to
very high [fascicle lengths: ICC 0.77 0.91, SEM 9 17
mm, and SEM% 8.79.7% (4, 29); pennation angles: ICC
0.88 0.97, SEM 1.0 1.3, and SEM% 8.8 9.5%] (4, 28,
29). Although the SEM of 17 mm appears very large, this was
for the semitendinosus muscle, which has very long muscle
fascicles; the SEM% was, therefore, 10% (29). As a result of
the small sample sizes in these cadaveric studies, the confi-
dence intervals for the ICC statistics were very wide, ranging
from 0.24 to 0.99 for fascicle lengths (4), and 0.23 to 0.99 (4)
and 0.62 to 1.00 for pennation angles (28). These three studies
imaged triceps brachii, biceps femoris, semitendinosus, and
medial gastrocnemius under relaxed and stationary conditions
(4, 28, 29).
Muscle fascicle lengths and pennation. The mean values of
muscle fascicle lengths and pennation angles are reported in
Tables 4 and 5. These mean values were obtained from both
anatomical inspection of cadaver muscles and ultrasound pro-
cedures and included values from supraspinatus, triceps
brachii, brachialis, extensor digitorum communis, vastus late-
J Appl Physiol doi:10.1152/japplphysiol.01430.2011 www.jappl.org
Kwah LK et al. 767
Ultrasound, Ref. Nos.
44
16 2 27
44
ralis, rectus femoris, biceps femoris, semitendinosus, soleus,
medial, and lateral gastrocnemius.
DISCUSSION
To our knowledge, this is the first systematic review of
studies of the reliability and validity of ultrasound measure-
ment of human muscle fascicle lengths and pennation. The data
show 2D ultrasound provides reliable measures of human
muscle fascicle lengths and pennation angles across a broad
range of experimental conditions. Studies on validity are few,
but the limited evidence suggests 2D ultrasound provides valid
measures of human muscle fascicle lengths and pennation
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angles, at least when large limb muscles are imaged in a
relaxed state with no joint or limb movement.
Ultrasound measurement of fascicle lengths and pennation
angles are reliable when muscles are imaged in relaxed and
contracted states, and when measurements are repeated be-
tween ultrasound sessions, images, and raters. Only a small
number of studies involved experienced radiologists/sonogra-
phers (Table 2), suggesting that reliable measurements are
possible without formal training in ultrasound imaging.
While the majority of studies reported high-reliability esti-
mates, two studies reported lower reliability estimates for
muscle fascicle lengths (SEM 17 mm, SEM% 14.2%) and
pennation angles (ICC 0.51 and CV 13.5%) (42, 56).
Several factors might explain the lower reliability observed in
these two studies. Muscle architecture could change between
ultrasound sessions held a few weeks apart. Inconsistent align-
ment of the probe (3, 61) could also contribute to inconsistency
of measurements of fascicle length and pennation angle.
Evidence of the validity of 2D ultrasound for measuring
muscle fascicle lengths and pennation in humans is limited.
Estimates obtained from three studies suggest that ultrasound
imaging has high to very high validity. However, the confi-
dence intervals about the point estimates were wide due to the
very small sample sizes in these studies (N 10) (4, 28, 29).
The finding of high validity can only be generalized to the
conditions employed in these three studies: most importantly,
the muscles were large limb muscles, and the muscles were
relaxed and stationary. Cadaveric studies cannot test the valid-
ity of measurements made on contracting muscles or under
moving conditions. More complete validation will require
development of new methods, such as the methods used by
Stark and Schilling (61) to compare muscle architecture in
small-animal muscles under relaxed and contracted states.
Data from cadaveric studies do not yet provide clear guid-
ance on how best to orientate the ultrasound probe when
measuring muscle fascicle lengths or pennation. If the probe is
not aligned with the plane of the muscle fascicles, muscle
768 Ultrasound Measurement of Human Muscle Fascicles
fascicle lengths and pennation angles could be underestimated
or overestimated (4). Most studies use one of two methods to
determine the alignment of the ultrasound probe with the plane
of muscle fascicles. One method involves positioning the probe
perpendicular to the skin (4, 6, 18, 28, 36, 47, 5254). The
other method involves adjusting probe alignment until image
quality is optimized (1, 4, 6, 10, 12, 13, 18, 27, 31, 48, 53, 54).
Two validity studies orientated the probe perpendicular to the
skin (4, 28). Data from these studies suggest the measurements
were reasonably valid. These limited data do not indicate how
well the probe is aligned with the plane of the fascicles when
the method of image optimization is used, or whether the
method of image optimization is better than positioning the
probe perpendicular to the skin.
We have considered two issues regarding the validity of 2D
ultrasound imaging in measuring muscle fascicle lengths and
pennation in humans: the difficulty in testing the validity of 2D
ultrasound imaging in contracted or moving muscles, and the
limited evidence for the best method of probe alignment.
Future studies on validity should consider these issues. The use
of 3D ultrasound imaging (25, 33), magnetic resonance imag-
ing (58), or diffusion tensor-magnetic resonance imaging (35,
59) might be a feasible alternative for a gold standard in
measuring muscle fascicle lengths and pennation instead of
anatomical inspection. This will make it easier to test the
validity of 2D ultrasound imaging in contracting muscles, since
cadavers will not be required. Validation studies comparing 3D
ultrasound imaging, magnetic resonance imaging, or diffusion
tensor-magnetic resonance imaging with anatomical inspection
measurements of muscle fascicle lengths and pennation may
have to be carried out first.
From the studies in our systematic review, we also extracted
mean values of fascicle lengths and pennation angles from
relaxed muscles measured in resting anatomical joint positions.
These data might be useful to physiologists who are interested in
constructing 3D models of human muscles. However, the data
from our systematic review are not a comprehensive compendium
of all of the published data on muscle fascicle lengths and
pennation in humans because we did not include data from papers
that reported measurements of human muscle fascicle lengths and
pennation but did not repeat their measurements or did not
compare their ultrasound measurements with cadaveric measure-
ments.
Conclusions. 2D ultrasound can provide reliable measure-
ments of human muscle fascicle length and pennation angle
across a broad range of experimental conditions. The limited
evidence suggests these measures are valid, at least when large
limb muscles are imaged in a relaxed state and there is no
movement of the limb or joint. Future studies on validity
should consider ways to test for the validity of 2D ultrasound
imaging in contracted or moving muscles and the best method
of probe alignment.
ACKNOWLEDGMENTS
The authors thank Min Jiat Teng for assistance with identification and
screening of studies.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
J Appl Physiol doi:10.1152/japplphysiol.01430.2011 www.jappl.org
Kwah LK et al.
AUTHOR CONTRIBUTIONS
Author contributions: L.K.K., R.Z.P., J.D., and R.D.H. conception and
design of research; L.K.K., R.Z.P., J.D., and R.D.H. analyzed data; L.K.K.,
R.Z.P., J.D., and R.D.H. interpreted results; L.K.K. and R.D.H. prepared
figures; L.K.K. and R.D.H. drafted manuscript; L.K.K., R.Z.P., J.D., and
R.D.H. edited and revised manuscript; L.K.K., R.Z.P., J.D., and R.D.H.
approved final version of manuscript.
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