473

ARTICLE
Examination of the confounding effect of subcutaneous fat on
muscle echo intensity utilizing exogenous fat
€ ller, Fabio J. Lanferdini, Juliana Y. Passos Karam, and Heiliane de Brito Fontana
Jonathan Neto Mu
Appl. Physiol. Nutr. Metab. Downloaded from cdnsciencepub.com by 191.191.70.62 on 05/25/21

Abstract: We aimed to provide an unbiased estimate of the confounding effect of subcutaneous fat thickness on ultrasound
echo intensity (EI) measures of muscle quality. The effect of fat thickness on EI was verified for an approximate range of
0 to 3 cm of fat using exogeneous layers of pork fat over the human tibialis anterior muscle. Sonograms were obtained
(i) with focus constant across fat thickness conditions, and (ii) with focus position adjusted to the muscle region of interest
(ROI) position for each fat thickness level. In agreement with our hypothesis, increasing fat between the probe and the ROI
resulted in a decrease in EI. This overestimating effect of fat on muscle quality differs between sonograms with constant
focus and sonograms with focus position adjusted to the vertical displacement in ROI position that occurs for different lev-
els of fat thickness. Correcting equations to account for the overestimating effect of fat on muscle quality are provided for
both focus conditions. This is the first study to systematically analyze the confounding effect of fat thickness as an inde-
pendent factor and the provided equations can be used for improved accuracy in estimates of muscle quality in obese/
overweight subjects/patients.
Novelty:

The independent confounding effect of subcutaneous fat thickness on ultrasound (US) estimates of muscle quality was
quantified.

US estimates of muscle quality depend on whether focus is adjusted to the muscle region of interest or not.
For personal use only.

Equations for correcting muscle quality estimates are provided.

Key words: fat, ultrasound, methods, agreement, focus, force, tissue, muscle quality, intramuscular fat, skeletal muscle.
Résumé : Notre but est de présenter une estimation non biaisée de l’effet de confusion de l’épaisseur de la graisse sous-cuta-
née sur les mesures de l’intensité de l’écho par ultrasons (« EI ») de la qualité musculaire. L’effet de l’épaisseur de la graisse
sur l’EI est vérifié pour un écart approximatif de 0 à 3 cm de graisse en utilisant des couches exogènes de graisse de porc sur
le muscle tibial antérieur humain. Des échographies sont obtenues (i) avec une mise au point constante du foyer dans
toutes les conditions d’épaisseur de graisse et (ii) avec l’ajustement de la mise au point du foyer à la région musculaire d’in-
térêt (« ROI ») pour chaque niveau d’épaisseur de graisse. Conformément à notre hypothèse, l’augmentation de l’épaisseur
de la graisse entre la sonde et la ROI suscite une diminution de l’EI. Cet effet de surestimation de la graisse sur la qualité
musculaire diffère entre les sonogrammes à mise au point constante du foyer et les sonogrammes avec la mise au point
ajustée au déplacement vertical de la ROI qui se produit pour différents niveaux d’épaisseur de graisse. Des équations de
correction pour tenir compte de l’effet de surestimation de la graisse sur la qualité musculaire sont fournies pour les deux
conditions de mise au point. Il s’agit de la première étude à analyser systématiquement l’effet de confusion de l’épaisseur
de la graisse en tant que facteur indépendant; les équations fournies peuvent être utilisées pour améliorer la précision des
estimations de la qualité musculaire chez les sujets/patients obèses/en surpoids. [Traduit par la Rédaction]
Les nouveautés :

L’effet de confusion indépendant de l’épaisseur de la graisse sous-cutanée sur les estimations de la qualité musculaire par
ultrasonographie (« US ») est quantifié.

Les estimations par ultrasonographie de la qualité musculaire dépendent de l’ajustement ou non du foyer à la région muscu-
laire d’intérêt.

Des équations pour corriger les estimations de la qualité musculaire sont fournies.

Mots-clés : graisse, échographie, méthodes, accord, foyer, force, tissu, qualité musculaire, graisse intramusculaire, muscle
squelettique.

Received 19 May 2020. Accepted 1 November 2020.

€ ller, F.J. Lanferdini, and J.Y. Passos Karam. Biomechanics Laboratory, School of Sports, Center of Sports, Federal University of Santa Catarina,
J. Neto Mu
Florianópolis, SC 88040-900, Brazil.
H. de Brito Fontana. Biomechanics Laboratory, School of Sports, Federal University of Santa Catarina, Florianópolis, Santa Catarina, Brazil; Department
of Morphological Sciences, School of Biological Sciences, Federal University of Santa Catarina, Florianópolis, Santa Catarina, Brazil.
Corresponding author: Heiliane de Brito Fontana (email: heiliane.fontana@ufsc.br).
Copyright remains with the author(s) or their institution(s). Permission for reuse (free in most cases) can be obtained from copyright.com.

Appl. Physiol. Nutr. Metab. 46: 473–478 (2021) dx.doi.org/10.1139/apnm-2020-0413 Published at www.nrcresearchpress.com/apnm on 6 November 2020.
 474
Introduction
Echo intensity (EI) of skeletal muscle sonograms has been used
as an estimate of muscle quality in individuals with various phe-
notypes (Blue et al. 2018; Herda et al. 2020; Mota and Stock 2017;
Stock et al. 2020), with greater EI scores indicating a greater accu-
mulation of intramuscular connective tissue and/or fat (Pillen
et al. 2009; Young et al. 2015). EI has been proved to be reliable
(Caresio et al. 2015; Lanferdini et al. 2019), thereby encouraging
its application in investigating training and aging adaptations
(Fukumoto et al. 2012a; Mota and Stock 2017).
Associations between subcutaneous fat thickness and EI esti-
mates of muscle quality in humans are usually discussed from
a physiological perspective (Herda et al. 2020; Oranchuk et al.
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2020; Ryan et al. 2016; Vakula et al. 2019), addressing the damag-
ing effect of obesity on muscle quality that has been confirmed
in animal models (Biltz et al. 2020; Collins et al. 2018). However,
the confounding effect that a fat layer may have on EI estimates
of muscle quality has not been carefully investigated and is cur-
rently not properly taken into account in the literature (Arts
et al. 2010; Nijboer-Oosterveld et al. 2011; Schless et al. 2019; Stock
et al. 2020).
The confounding effect of subcutaneous fat thickness on EI
estimates of muscle quality leads to an underestimation of the neg-
ative impact of obesity/overweight on muscle health (Oranchuk
et al. 2020; Ryan et al. 2016) and may lead to erroneous conclusions
regarding chronic exercise-induced changes in muscle quality
(Wong et al. 2020) and differences in muscle quality between men
and women or between muscles and muscle regions (Oranchuk
For personal use only.
et al. 2020; Stock et al. 2020). Until now, most studies that correct
ultrasound-based estimates of muscle quality for the confounding
effect of subcutaneous fat have counted on the equations devel-
oped by Young et al. (2015).
Young et al. (2015) proposed a correction factor for subcutane-
ous fat thickness on EI estimates of muscle quality that has been
widely used (Blue et al. 2018; Ryan et al. 2016; Stock et al. 2020).
However, their experiment consisted on changing fat thickness
by applying different levels of external pressure on the skin,
which is expected to lead to changes in tissue parameters, such
as density and stiffness, that influence the speed of ultrasound
(US) wave propagation (Kot et al. 2012; Winn et al. 2016), produc-
ing changes in EI that may not represent the effect of subcutane-
ous fat thickness (Ishida et al. 2016). The authors, therefore, did
not evaluate the independent effect of fat but only the associa-
tion between changes in fat thickness and changes in EI that
occur when pressure is applied to the skin. Additionally, their
correction factor was incorrectly based on average estimates of
EI scores for a thickness of 1 cm, instead of on the slope of the
relationship between EI and thickness. This methodological issue
results in further limitations in the use of the equation proposed,
especially when extrapolated to other subjects/muscles, as their
factor essentially reflects an (compression-biased) estimate of the
quality of the muscle samples analyzed and not the effect of subcu-
taneous fat.
Despite the appeal of ultrasound EI as a noninvasive, low-cost
surrogate measure of muscle quality and lipid accumulation
within skeletal muscle (Choi et al. 2016), estimates depend on the
standardization of ultrasound parameters to be reliable (Caresio
et al. 2015; Paris et al. 2020). Most studies standardize gain and
time gain compensation (TGC), and recently the importance of
maintaining depth fixed across sonogram acquisitions has been
systematically demonstrated (Paris et al. 2020). However, for com-
parisons made across individuals with different subcutaneous fat
thickness; fixing gain, time gain compensation and depth still
does not control for changes in the relative position between the
region of interest (ROI) and the sonogram focus position. Muscle
ROI position within the sonogram window is displaced down-
wards as the amount of subcutaneous fat in the sonogram
Appl. Physiol. Nutr. Metab. Vol. 46, 2021
increases. Focus position, in contrast is usually set automatically
to a fixed proportion of depth. Therefore, if focus is not adjusted
manually, the vertical distance between focus and the muscle
ROI positions will vary depending on fat thickness. This effect
has not yet been analyzed and is not accounted in the current
methods used to correct EI estimates of muscle quality for the
confounding effect of fat thickness.
In this study, we aim to provide an unbiased estimate of the
effect of subcutaneous fat thickness on EI scores. Fat thickness
was modified by adding exogenous fat layers over the skin, and the
resulting change in EI was analyzed. This experimental approach
allowed us to mimic the effects of increasing fat thickness (attenua-
tion and changes in ROI relative position) on EI estimates of muscle
quality. We hypothesized that increasing fat thickness between the
probe and the ROI in the muscle leads to an overestimation of mus-
cle quality. To explore the interactions between the effect of fat and
the vertical displacement of ROI relative to the sonogram focus, we
compared EI measurements obtained with and without adjusting
the focus to the ROI position for each fat thickness level. Correcting
equations for the effect of fat thickness are proposed for sono-
grams with and without focus adjustment.
Materials and methods
Ten (5 males and 5 females) healthy subjects (age, 28.0 6
5.6 years; height, 1.70 6 0.11 m; body mass, 67.8 6 11.9 kg) partici-
pated in this study. Participants were selected based on convenience
and gave full written informed consent to participate in the study.
This study was conducted according to the Declaration of Helsinki, and
the procedures were approved by the local Institutional Research
Ethics Committee of the Federal University of Santa Catarina
(1.721.566). The effect of fat thickness on EI estimates of muscle qual-
ity was evaluated for an approximate range of 0 to 3 cm of fat using
the tibialis anterior (TA) muscle and exogeneous fat layers. Six layers
of pork fat (layers 1 to 6) with approximate length, width and thick-
ness of 8  4  0.5 cm were used.
All participants were instructed to maintain their normal rou-
tine and avoid strenuous exercise in the 48 h before the data col-
lection. Before measurements, participants rested supine on a
stretcher for 5 min. To identify TA muscle belly limits, a 50 mm
linear array ultrasound probe (8 MHz) was positioned at the mid-
distance between the lateral malleolus and the lateral knee con-
dyle and a transversal ultrasound B-mode scan was performed
(LOGIC S7 Expert, General Electric, Wauwatosa, USA). Sonograms
were then taken from the mid-belly with the ultrasound probe longi-
tudinally aligned with the muscle and perpendicular to the skin.
The probe was coated with water-soluble transmission gel and
care was taken to avoid compression of the skin. The skin was
shaved to remove the excess of hair and avoid a possible unknown con-
founding effect of hair in the gel interface. B-Mode was configured with
an image depth of 40 mm; general gain of 60 dB and TGC at neutral
position. Two sonograms were obtained with the probe directly over
the skin (layer 0) and for each additional exogenous layer (layers 1 to 6);
one in which the focus was kept at 1 cm (without focus adjustment)
and another in which the focus was adjusted (with focus adjustment)
to coincide with the ROI (area right below TA superficial aponeurosis).
After obtaining the 2 sonograms for layer 6, the fat layers were
removed from the skin and the procedure was repeated twice,
resulting in 3 sonograms for each layer and focus condition.
Sonogram analysis was conducted by 2 evaluators using ImageJ
1.42q software (National Institute of Health, Bethesda, USA) and
included measurements of adipose layer thickness (AT) and mus-
cle EI. Excellent agreement was reached between evaluators
(intraclass correlation coefficient (ICC) (2,1) >0.934 for all meas-
urements and conditions).
AT was defined as the distance between the TA superficial apo-
neurosis and the superficial limit of the adipose layer measured.
EI was determined in the ROI using the mean value of a standard
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 € ller et al.
Neto Mu
Fig. 1. Sonogram (left) including the muscle region of interest (ROI) and the associated schematic representation (right) showing the
subcutaneous fat layer and the 6 fat tissue layers used in our experiment. EI, echo intensity.
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For personal use only.
gray-scale histogram function, with values ranging between 0 (black)
and 255 (white). The ROI consisted of a rectangle (4.0  1.0 cm) posi-
tioned below the superficial aponeurosis of the TA as shown in Fig. 1.
Statistical analysis
The mean across the 3 repeated sonograms for each condition
(layer  focus adjustment) per subject was used. Precision for
repeated procedures yielded ICC (2,3) values greater than 0.84
(p < 0.001) for EI and greater than 0.953 (p < 0.001) for AT in all condi-
tions. Normal distribution and sphericity of EI and adipose tissue data
were verified using Shapiro–Wilk and Mauchly tests, respectively.
Intraclass correlation (ICC 2,1  agreement) was tested between
focus adjustment conditions with ICC ≤ 0.25 being classified as
absent correlation; 0.25 < ICC ≤ 0.5, as weak correlation; 0.5 <
ICC ≤ 0.7, as moderate; 0.7 < ICC ≤ 0.9, as strong; and ICC > 0.9 as
very strong (Caresio et al. 2015). Standard error of measurement
(SEM) and minimum detectable change (MDC) were calculated
using eqs. (1) and (2). Confidence interval was set at 95% and
z score adjusted to n = 10.
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ð1Þ SEM ¼ SDpooled  1  ICC
pffiffiffi
ð2Þ MDC95 ¼ SEM  2:228  2 the condition (Table 1). Agreement between EI scores obtained with
For subjects pooled data, the effect of increasing adipose tissue
thickness on EI scores was best-fitted using linear regression
analysis for the focus-adjusted condition and was best fitted using
quadratic regression analysis for the without focus-adjusted
condition. Based on the effect observed, correction equations were
475
developed for sonograms obtained with and without focus adjust-
ment separately. The correcting equations were based on the subtrac-
tion of the confounding effect of AT on EI scores (ATeffect) from a
measured EI value.
EIcorrected ¼ EImeasured  ATeffect
To estimate ATeffect, the effect of AT on EI was calculated for ev-
ery participant using individual best-fit regression equations.
Quadratic regressions were used to best fit the effect of fat on
sonograms obtained without focus adjustment, while linear
regression equations were used to best fit the effect of fat on sono-
grams obtained with focus adjustment. Finally, the average effect
(ATeffect) was calculated by averaging the regression coefficients
across participants. Standard errors were calculated for each coeffi-
cient of the correcting equations. For all analyses, GraphPad Prism
(USA) was used with a significance level set a = 0.05.
Results
AT varied from approximately 0.4 to 3.0 cm with the additional fat
layers, while EI scores varied from 138 6 11 to 43 6 17 depending on
and without focus adjustment was highly dependent on AT. While a
very strong ICC and a SEM <1% were observed for sonograms with a
thin fat layer, for sonograms accompanied with a fat layer thicker
than 2 cm, ICC between sonograms with and without focus adjust-
ment decreased to weak or absent correlation with SEM values
reaching values greater than 30% when all 6 layers were included in
the analysis (Table 1 – agreement analysis).
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 476 Appl. Physiol. Nutr. Metab. Vol. 46, 2021

Table 1. Mean (SD) of echo intensity (EI) scores and adipose thickness (AT) (n = 10) for an increasing number of adipose layers.
EI (a.u.) Agreement between focus conditions (1  2)
No. of layers AT, cm* 1. Focus not adjusted 2. Focus adjusted ICC [95%CI] F SEM% MDC
0† 0.40 (0.10) 137.3 (11.2) 137.7 (12.5) 0.988 [0.955, 0.997] 158.945‡ 0.9 4.0
1 0.87 (0.12) 122.1 (12.4) 123.0 (13.5) 0.953 [0.832, 0.988] 39.919‡ 2.2 8.6
2 1.28 (0.12) 105.2 (18.8) 104.8 (18.2) 0.979 [0.917, 0.995] 83.981‡ 2.5 8.2
3 1.63 (0.09) 99.6 (21.5) 85.9 (23.8) 0.826 [–0.041, 0.969] 78.246‡ 10.4 30.5
4 1.92 (0.14) 98.1 (18.4) 75.5 (20.2) 0.561 [–0.037, 0.898] 32.598‡ 16.9 46.1
5 2.41 (0.17) 87.2 (13.2) 57.1 (18.3) 0.327 [–0.024, 0.774] 20.244‡ 24.9 56.7
6 2.95 (0.17) 76.9 (9.5) 42.9 (16.6) 0.160 [–0.041, 0.575] 5.408§ 33.1 62.5
Note: Values were obtained from sonograms with and without focus adjustment. Estimates of agreement for EI between procedures are shown: intraclass
correlation coefficient (ICC [2,1] agreement) with confidence intervals (CI), standard error of measurement (SEM), and minimal detectable changes (MDC).
*Mean across focus conditions (average agreement ICC [2,2] = 0.99).
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†
No additional adipose layers, only the subject’ subcutaneous fat.
‡
p = 0.001.
§
p = 0.01.

Fig. 2. Regression analysis between echo intensity scores and varying adipose layer thickness. (A) Pooled raw data (n = 10) obtained from
sonograms with focus fixed at 1 cm, and (B) pooled raw data obtained from sonograms with the focus adjusted to the region of interest.
Note that echo intensity decreases with increasing adipose layer thickness, even though the muscle (and its “quality”) is not changed for
a given subject.
For personal use only.

For both focus conditions, a significant effect of AT was observed
on EI, with EI scores decreasing as thickness increased (p < 0.001).
While a quadratic relationship was observed between EI and AT
for sonograms acquired without focus adjustment (Fig. 2A), the
relationship between EI and AT for sonograms with the focus
adjusted to the ROI was best fit with a linear equation (Fig. 2B).
A stronger prediction of AT effect on EI was obtained for sono-
grams obtained with focus adjustment compared with those
without adjustment. While 69% of the variability in EI was associ-
ated with changes in AT in sonograms with focus adjustment
(Fig. 2B), maintaining the focus fixed at 1 cm (Fig. 2A) allowed
only for 54% of the variability to be explained using a second
order polynomial equation.
Equations for correcting for the effect of AT on EI estimates of
muscle quality are presented below for sonograms without focus
adjustment (i) and with focus adjustment (ii).
ðiÞ EIcorrected ¼ EImeasured  5:0054  AT2 þ 38:30836  AT
ðiiÞ EIcorrected ¼ EImeasured þ 39:2297  AT
where standard errors of coefficients in eq. (i) were 62.24 and 68.27,
respectively, and, in eq. (ii) 62.09.
Discussion
Skeletal muscles produce the forces required for movement
and play an important role in regulating body metabolism (Baskin
et al. 2015; Dickinson et al. 2000). Muscle quality is an important de-
terminant of muscle function (Goodpaster et al. 2006; MacLennan
et al. 2020), and can be estimated from ultrasound EI assessment
in a noninvasive, easily accessible, and relatively inexpensive man-
ner (Choi et al. 2016; Watanabe et al. 2018; Young et al. 2015). Given
the popularity of EI in clinical settings, it is crucial to maximize its
accuracy (Heymsfield et al. 2015).
Every 1-cm increase in AT may result in an underestimation of
EI of more than 39 arbitrary units on a scale from 0 to 255. Consid-
ering that subcutaneous fat thickness varies substantially across
individuals and across muscles and muscle regions (Oranchuk
et al. 2020; Stock et al. 2020), estimates of muscle quality that are
not corrected for this effect are biased. More importantly, mus-
cle quality is often considered in people with obesity undergoing
weight gain/loss and nutritional interventions (Blue et al. 2018;
Rahemi et al. 2015), and for those, the confounding effect of AT
on muscle quality estimates will become quite substantial due to
the large amounts of subcutaneous fat.
If no correction is applied, differences in muscle quality
between subjects with different subcutaneous fat are underesti-
mated: while a worst muscle quality – i.e., greater EI – may be
expected in individuals with greater fat accumulation (Choi et al.
2016; Ponti et al. 2019), the confounding effect of subcutaneous
fat on EI will reduce EI units, leading to similar estimated values
of muscle quality as for a thin person with good muscle quality.
This confounding effect may also explain weak associations between
EI and AT or percentage of body fat (Fukumoto et al. 2012a), and
between EI and functional parameters (Stock et al. 2018).
Despite early studies pointing to the need of considering sub-
cutaneous fat thickness for diagnostic evaluation of muscle echo-
genicity (Blue et al. 2018; Reimers et al. 1993), this issue is still
disregarded in the growing literature on EI and muscle quality.

Published by NRC Research Press

 € ller et al.
Neto Mu
Only recently, Young et al. (2015) developed a correcting equation
for the effect of subcutaneuos fat on ultrasound-based estimates
of muscle quality and their equation has been used widely in the
literature. However, the independent effect of fat thickness was
not evaluated in their study but only how the changes in fat
thickness are associated to the changes in EI when pressure is
applied to the skin. Additionaly, as indicated in the introduction,
the procedures used to derive the equation are rather unsound
as the correction factor proposed was the absolute estimated
value of EI measured in their sample (5 participants, muscle not
informed) for a subcutaneous fat thickness of 1 cm (EI  40). By
doing so, they introduced an arbitrary correction factor that does
not represent “the addition of EI with every 1.0-cm subcutaneous
fat thickness” as claimed but solely the estimated quality of the
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muscles analyzed for a subcutaneous fat thickness of 1 cm. When
using their equation, therefore, one has to assume that the esti-
mated EI of the muscles evaluated in their experiment (muscle
not specified) for a subcutaneous thickness of 1 cm can be used as
a linear factor that represents the changes in EI with increasing
fat thickness. To truly evaluate the rate of change in EI, the slope
of the relationship between subcutaneous fat thickness and EI
should have been used as the correction factor.
The slope of the relationship between subcutaneous fat thick-
ness and EI observed by Young et al. (2015) with tissue compres-
sion (fig. 1 in their manuscript) differs substantially from the one
observed in our study (Fig. 2A). Differences may be explained by
the nature of the experiment conducted by Young et al. (2015),
which does not allow for the quatification of the independent
effect of fat thickness, and also by the the limited range of fat thick-
For personal use only.
ness tested in their experiment. Only 2 out of the 5 participants
tested presented subcutaneous fat thickness greater than 1 cm and
changes in subcutaneous fat thickness yielded by compression
were very small (0.4 cm). With our experimental approach, we
were able to assess changes in AT by an average of 2.95 6 0.17 cm,
which allowed us to quantify the confounding effect of fat with-
out having to extrapolate the data assuming a linear effect.
Adjusting the focus to the muscle ROI optimizes the image by
increasing the spatial resolution for a specific area. Although this
approach has been previously used for measuring EI estimates of
muscle quality (Fukumoto et al. 2012b; Masaki et al. 2016; Taniguchi
et al. 2017), most often focus position is not specified in the litera-
ture. The confounding effect of fat thickness is different between
sonograms obtained with a fixed focus position (Fig. 2A) and sono-
grams obtained with the focus adjusted to the ROI position within
the sonogram (Fig. 2B). Therefore, it is important that the equations
proposed in or study are used accordingly. As the thickness of the
subcutaneous fat layer increases, and the ROI is displaced down-
wards in the sonogram window, measurements with and without
focus adjustment become poorly related (Table 1) with errors reach-
ing 33%. The confounding effect of changes in the distance between
muscle ROI and focus position on the equations proposed by
Young et al. (2015) are not known as focus was set to maximum
and depth was changed freely across participants to accommo-
date fat thickness.
In this study, we experimentally manipulated the thickness of
the fat layer between the probe and the muscle region of interest,
which has allowed us to quantify the cofounding effect of subcu-
taneous fat on EI estimates of muscle quality. The TA muscle was
used as a model due to the thin subcutaneous fat associated,
which has allowed us to manipulate fat thickness across a wide
range of values. An average effect was identified across subjects
(men and women), with approximately 70% and 55% of total vari-
ability in EI (sonograms with and without focus adjustment,
respectively) explained by the changes in AT. To account for this
confounding effect of fat on EI estimates, future studies may
choose to use the equations proposed in this study. While we can-
not be sure this effect is similar for other muscles or for individuals
with different phenotypes, the relatively small variation across
477
subjects in this study suggest that interindividual variability
with regards to the effect of fat are small when compared with
the magnitude of the average confounding effect observed.
Longitudinal sonograms were used as they allow for the simul-
taneous determination of muscle architectural parameters, such
as physiological cross-sectional area, fascicle length and penna-
tion angle that are often of interest in studies evaluating muscle
quality and are not possible to estimate using transversal scans.
While the independent effect of fat thickness is not expected to
change between transversal and longitudinal scans, this compar-
ison would require further investigation.
Acute and chronic changes in EI have been observed with exer-
cise. However, further experimental work is needed to provide
appropriate interpretations of these changes in physiological
and clinical contexts (Routledge et al. 2019; Rowe et al. 2019;
Wong et al. 2020). The omission and/or inappropriate accounting
of the effects of subcutaneous fat on EI might partly explain
inconsistent results in skeletal muscle tissue quality following
resistance training reported in a recent review (Wong et al.
2020).
In our experiment, pork fat was used as a surrogate for human
fat. The choice of using pork fat was based on the ease and safety
of access to this material. In this experiment we assume the
effect of pork fat is similar to human fat, but it is possible that dif-
ferences exist. Given the similarities between the subjects’ sub-
cutaneous layer and the added layers on the ecogram, we believe
US waves behave similarly in pork fat and in human fat. Future
studies investigating this issue in cadavers will add to our current
knowledge regarding the confounding effect of fat on ultra-
sound-based estimates of muscle quality.
Conclusion
We conclude from the results of this study that (i) subcutane-
ous fat affects muscle quality estimates based on EI of ultrasound
imaging; (ii) that focus adjustment is crucial in quantitative
assessments of muscle quality by EI; and (iii) that the correcting
equations proposed here prevent the overestimation of muscle
quality in people/muscles with thick subcutaneous fat layers.
Conflict of interest statement
The authors have no conflict of interest to report.
Acknowledgements
We acknowledge The National Council of Scientific and
Technological Development (CNPq), an agency of the Ministry
of Science, Technology, Innovations and Communications (MCTIC)
in Brazil, for the scholarship (PIBIC) provided to J.N.M.
References
Arts, I.M., Pillen, S., Schelhaas, H.J., Overeem, S., and Zwarts, M.J. 2010.
Normal values for quantitative muscle ultrasonography in adults. Mus-
cle Nerve, 41(1): 32–41. doi:10.1002/mus.21458. PMID:19722256.
Baskin, K.K., Winders, B.R., and Olson, E.N. 2015. Muscle as a “mediator” of
systemic metabolism. Cell Metab. 21(2): 237–248. doi:10.1016/j.cmet.2014.
12.021. PMID:25651178.
Biltz, N.K., Collins, K.H., Shen, K.C., Schwartz, K., Harris, C.A., and Meyer, G.A.
2020. Infiltration of intramuscular adipose tissue impairs skeletal mus-
cle contraction. J. Physiol. 598(13): 2669–2683. doi:10.1113/JP279595. PMID:
32358797.
Blue, M.N.M., Smith-Ryan, A.E., Trexler, E.T., and Hirsch, K.R. 2018. The
effects of high intensity interval training on muscle size and quality in
overweight and obese adults. J. Sci. Med. Sport. 21(2): 207–212. doi:10.1016/
j.jsams.2017.06.001. PMID:28647284.
Caresio, C., Molinari, F., Emanuel, G., and Minetto, M.A. 2015. Muscle echo
intensity: reliability and conditioning factors. Clin. Physiol. Funct. Imaging,
35(5): 393–403. doi:10.1111/cpf.12175. PMID:24902991.
Choi, S.J., Files, D.C., Zhang, T., Wang, Z.M., Messi, M.L., Gregory, H., et al.
2016. Intramyocellular lipid and impaired myofiber contraction in nor-
mal weight and obese older adults. J. Gerontol. A Biol. Sci. Med. Sci. 71(4):
557–564. doi:10.1093/gerona/glv169. PMID:26405061.
Published by NRC Research Press
 478
Collins, K.H., Herzog, W., MacDonald, G.Z., Reimer, R.A., Rios, J.L., Smith, I.C.,
et al. 2018. Obesity, metabolic syndrome, and musculoskeletal disease: com-
mon inflammatory pathways suggest a central role for loss of muscle integ-
rity. Front. Physiol. 9: 112. doi:10.3389/fphys.2018.00112. PMID:29527173.
Dickinson, M.H., Farley, C.T., Full, R.J., Koehl, M.A., Kram, R., and Lehman, S.
2000. How animals move: an integrative view. Science, 288(5463): 100–106.
doi:10.1126/science.288.5463.100. PMID:10753108.
Fukumoto, Y., Ikezoe, T., Yamada, Y., Tsukagoshi, R., Nakamura, M.,
Mori, N., et al. 2012a. Skeletal muscle quality assessed from echo inten-
sity is associated with muscle strength of middle-aged and elderly per-
sons. Euro. J. Appl. Physiol. 112(4): 1519–1525. doi:10.1007/s00421-011-2099-5.
PMID:21847576.
Fukumoto, Y., Ikezoe, T., Tateuchi, H., Tsukagoshi, R., Akiyama, H., So, K.,
et al. 2012b. Muscle mass and composition of the hip, thigh and abdomi-
nal muscles in women with and without hip osteoarthritis. Ultrasound
Med. Biol. 38(9): 1540–1545. doi:10.1016/j.ultrasmedbio.2012.04.016. PMID:
22749818.
Appl. Physiol. Nutr. Metab. Downloaded from cdnsciencepub.com by 191.191.70.62 on 05/25/21
Goodpaster, B.H., Park, S.W., Harris, T.B., Kritchevsky, S.B., Nevitt, M.,
Schwartz, A.V., et al. 2006. The loss of skeletal muscle strength, mass,
and quality in older adults: the health, aging and body composition
study. J. Gerontol. A Biol. Sci. Med. Sci. 61(10): 1059–1064. doi:10.1093/gerona/
61.10.1059. PMID:17077199.
Herda, T.J., Ryan, E.D., Kohlmeier, M., Trevino, M.A., Gerstner, G.R.,
Roelofs, E.J., et al. 2020. Muscle cross-sectional area and motor unit prop-
erties of the medial gastrocnemius and vastus lateralis in normal weight
and overfat children. Exp. Physiol. 105(2): 335–346. doi:10.1113/EP088181.
PMID:31785108.
Heymsfield, S.B., Gonzalez, M.C., Lu, J., Jia, G., and Zheng, J. 2015. Skeletal
muscle mass and quality: evolution of modern measurement concepts in
the context of sarcopenia. Proc. Nutr. Soc. 74(4): 355–366. doi:10.1017/
S0029665115000129. PMID:25851205.
Ishida, H., Suehiro, T., and Watanabe, S. 2016. Influence of inward pressure
of the transducer on thickness and echo intensity of the rectus femoris
muscle during ultrasonography. Middle East J. Rehabil. Health Stud. 3(2):
e36059. doi:10.17795/mejrh-36059.
For personal use only.
Kot, B.C., Zhang, Z.J., Lee, A.W., Leung, V.Y., and Fu, S.N. 2012. Elastic modu-
lus of muscle and tendon with shear wave ultrasound elastography: var-
iations with different technical settings. PLoS One, 7(8): e44348. doi:10.
1371/journal.pone.0044348. PMID:22952961.
Lanferdini, F.J., Manganelli, B.F., Lopez, P., Klein, K.D., Cadore, E.L., and
Vaz, M.A. 2019. Echo Intensity Reliability for the Analysis of Different
Muscle Areas in Athletes. J Strength Cond. Res. 33(12): 3353–3360. doi:10.
1519/JSC.0000000000003063. PMID:31765345.
MacLennan, R.J., Sahebi, M., Becker, N., Davis, E., Garcia, J.M., and Stock, M.S.
2020. Declines in skeletal muscle quality vs. size following two weeks of
knee joint immobilization. PeerJ, 8: e8224. doi:10.7717/peerj.8224. PMID:
31976172.
Masaki, M., Ikezoe, T., Fukumoto, Y., Minami, S., Aoyama, J., Ibuki, S., et al.
2016. Association of walking speed with sagittal spinal alignment, muscle
thickness, and echo intensity of lumbar back muscles in middle-aged and
elderly women. Aging Clin. Exp. Res. 28(3): 429–434. doi:10.1007/s40520-015-
0442-0. PMID:26319656.
Mota, J.A., and Stock, M.S. 2017. Rectus Femoris Echo Intensity Correlates
with Muscle Strength, but Not Endurance, in Younger and Older Men.
Ultrasound Med Biol. 43(8): 1651–1657. doi:10.1016/j.ultrasmedbio.2017.04.010.
PMID:28533003.
Nijboer-Oosterveld, J., Van Alfen, N., and Pillen, S. 2011. New normal values
for quantitative muscle ultrasound: obesity increases muscle echo inten-
sity. Muscle Nerve. 43(1): 142–143. doi:10.1002/mus.21866. PMID:21082696.
Oranchuk, D.J., Stock, M.S., Nelson, A.R., Storey, A.G., and Cronin, J.B. 2020.
Variability of regional quadriceps echo intensity in active young men
with and without subcutaneous fat correction. Appl. Physiol. Nutr. Metab.
45(7): 745–752. doi:10.1139/apnm-2019-0601. PMID:31917597.
Appl. Physiol. Nutr. Metab. Vol. 46, 2021
Paris, M.T., Bell, K.E., Avrutin, E., and Mourtzakis, M. 2020. Ultrasound
image resolution influences analysis of skeletal muscle composition. Clin.
Physiol. Funct. Imaging, 40(4): 277–283. doi:10.1111/cpf.12636. PMID:32342635.
Pillen, S., Tak, R.O., Zwarts, M.J., Lammens, M.M., Verrijp, K.N., Arts, I.M.,
et al. 2009. Skeletal muscle ultrasound: correlation between fibrous tis-
sue and echo intensity. Ultrasound Med. Biol. 35(3): 443–446. doi:10.1016/
j.ultrasmedbio.2008.09.016. PMID:19081667.
Ponti, F., Santoro, A., Mercatelli, D., Gasperini, C., Conte, M., Martucci, M.,
et al. 2019. Aging and imaging assessment of body composition: from fat to
facts. Front Endocrinol. 10: 861. doi:10.3389/fendo.2019.00861. PMID:31993018.
Rahemi, H., Nigam, N., and Wakeling, J.M. 2015. The effect of intramuscular
fat on skeletal muscle mechanics: implications for the elderly and obese. J. R.
Soc. Interface, 12(109): 20150365. doi:10.1098/rsif.2015.0365. PMID:26156300.
Reimers, K., Reimers, C.D., Wagner, S., Paetzke, I., and Pongratz, D.E. 1993.
Skeletal muscle sonography: a correlative study of echogenicity and mor-
phology. J. Ultrasound Med. 12(2): 73–77. doi:10.7863/jum.1993.12.2.73. PMID:
8468739.
Routledge, H.E., Bradley, W.J., Shepherd, S.O., Cocks, M., Erskine, R.M.,
Close, G.L., et al. 2019. Ultrasound Does Not Detect Acute Changes in Gly-
cogen in Vastus Lateralis of Man. Med. Sci. Sports Exerc. 51(11): 2286–
2293. doi:10.1249/MSS.0000000000002052. PMID:31169793.
Rowe, G.S., Blazevich, A.J., and Haff, G.G. 2019. pQCT- and ultrasound-based
muscle and fat estimate errors after resistance exercise. Med. Sci. Sports
Exerc. 51(5): 1022–1031. doi:10.1249/MSS.0000000000001873. PMID:30986813.
Ryan, E.D., Shea, N.W., Gerstner, G.R., Barnette, T.J., Tweedell, A.J., and
Kleinberg, C.R. 2016. The influence of subcutaneous fat on the relation-
ship between body composition and ultrasound-derived muscle quality.
Appl. Physiol. Nutr. Metab. 41(10): 1104–1107. doi:10.1139/apnm-2016-0238.
PMID:27690567.
Schless, S.H., Cenni, F., Bar-On, L., Hanssen, B., Kalkman, B., O’Brien, T.,
et al. 2019. Medial gastrocnemius volume and echo-intensity after botuli-
num neurotoxin A interventions in children with spastic cerebral palsy.
Dev. Med. Child Neurol. 61(7): 783–790. doi:10.1111/dmcn.14056. PMID:30320442.
Stock, M.S., Oranchuk, D.J., Burton, A.M., and Phan, D.C. 2020. Age, sex, and
region-specific differences in skeletal muscle size and quality. Appl. Phys-
iol. Nutr. Metab. doi:10.1139/apnm-2020-0114. PMID:32450045.
Stock, M.S., Whitson, M., Burton, A.M., Dawson, N.T., Sobolewski, E.J., and
Thompson, B.J. 2018. Echo intensity versus muscle function correlations
in older adults are influenced by subcutaneous fat thickness. Ultrasound
Med. Biol. 44(8): 1597–1605. doi:10.1016/j.ultrasmedbio.2018.04.009. PMID:
29776601.
Taniguchi, M., Yamada, Y., Fukumoto, Y., Sawano, S., Minami, S., Ikezoe, T.,
et al. 2017. Increase in echo intensity and extracellular-to-intracellular
water ratio is independently associated with muscle weakness in elderly
women. Eur. J. Appl. Physiol. 117(10): 2001–2007. doi:10.1007/s00421-017-
3686-x. PMID:28755131.
Vakula, M.N., Fisher, K.L., Garcia, S.A., Holmes, S.C., Post, B.K., Costa, P.B.,
et al. 2019. Quadriceps impairment is associated with gait mechanics in
young adults with obesity. Med. Sci. Sports Exerc. 51(5): 951–961. doi:10.
1249/MSS.0000000000001891. PMID:30629047.
Watanabe, Y., Ikenaga, M., Yoshimura, E., Yamada, Y., and Kimura, M. 2018.
Association between echo intensity and attenuation of skeletal muscle in
young and older adults: a comparison between ultrasonography and
computed tomography. Clin. Interv. Aging, 13: 1871–1878. doi:10.2147/CIA.
S173372.
Winn, N., Lalam, R., and Cassar-Pullicino, V. 2016. Sonoelastography in the
musculoskeletal system: current role and future directions. World J.
Radiol. 8(11): 868–879. doi:10.4329/wjr.v8.i11.868. PMID:27928468.
Wong, V., Spitz, R.W., Bell, Z.W., Viana, R.B., Chatakondi, R.N., Abe, T.,
et al. 2020. Exercise induced changes in echo intensity within the muscle:
a brief review. J Ultrasound. 23: 457–472. doi:10.1007/s40477-019-00424-y.
PMID:31925731.
Young, H.J., Jenkins, N.T., Zhao, Q., and McCully, K.K. 2015. Measurement of
intramuscular fat by muscle echo intensity. Muscle Nerve. 52(6): 963–971.
doi:10.1002/mus.24656. PMID:25787260.
Published by NRC Research Press