Effect of prolonged sitting immobility on shear wave velocity of the lower leg muscles in healthy adults: A proof-of-concept study

Kumiko Okino; Mitsuhiro Aoki; Masahiro Yamane; Chikashi Kohmura

doi:10.1371/journal.pone.0251532

. 2021 May 10;16(5):e0251532. doi: 10.1371/journal.pone.0251532

Effect of prolonged sitting immobility on shear wave velocity of the lower leg muscles in healthy adults: A proof-of-concept study

Kumiko Okino ^1,^#, Mitsuhiro Aoki ^2,^*, Masahiro Yamane ^3,^#, Chikashi Kohmura ^1,^#

Editor: Guy Cloutier⁴

PMCID: PMC8109794 PMID: 33970970

Abstract

Objective

The purpose of this study is to investigate the physical changes of the lower leg muscles in the compartment by observing the changes in the shear wave velocity of the gastrocnemius, soleus and tibialis anterior muscles with time in the sitting position for 2 hours and after elevation of the lower leg.

Materials and methods

The subjects were 24 healthy adult males (average age 26.6 years). Shear wave velocity was measured by Aplio 500 in immobilized leg immediately after the start of sitting, 60 minutes and 120 minutes after the start of sitting. After 120 minutes the subjects raised the lower leg for 3 minutes, then measured again.

Results

In the lateral and medial gastrocnemius, there was a significant increase in the velocity at 60 (1.58 ± 0.06, 1.70 ± 0.09 m/s) and 120 minutes (1.70 ± 0.10, 1.83 ± 0.11 m/s) after the start of the test (1.52 ± 0.06, 1.66 ± 0.10 m/s), respectively (p<0.01). In the soleus and the tibialis anterior, there was a significant increase in the velocity at 120 minutes (1.89 ± 0.17, 2.30 ± 0.24 m/s) compared to after the start (1.60 ± 0.15, 2.15 ± 0.26 m/s), respectively (p<0.01). In all muscles, there was a significant decrease in the velocity after the raising compared to that of 120 minutes (p<0.01).

Conclusions

It has been reported that the change of shear wave velocity with time is proportional to the intramuscular pressure in the leg compartment, and it is assumed that the increase of shear wave velocity in the 2-hour seated leg is due to fluid retention in extra-cellular space of the compartment.

Introduction

In recent years, advances in shear wave elastography have made it possible to evaluate the mechanical properties (elastic modulus) of skeletal muscles in real time by measuring shear wave velocity. It is possible to measure the conditions of stretched muscles (static mechanical properties) and that of active muscle contraction (dynamic mechanical properties) [1, 2].

The measurement of elastic modulus of skeletal muscle began around 2005, and a series of reports by Gennisson et al. (2005, 2010) established that the elastic modulus of muscle can be accurately measured by placing a probe parallel to the muscle fibers [1, 3]. This has been confirmed by subsequent studies, which have shown that shear modulus measurements reflect the physical properties associated with muscle extension and contraction. At the same time, they evaluated the difference between anisotropy and isotropy of ultra-sound transmission brought about by transverse and longitudinal probe placements. Longitudinal measurements have become the most common way to measure the elastic modulus of muscle tissue in recent years [4].

Elastic modulus measurements of the triceps surae muscle in resting condition were initiated by Lacourpaille et al. (2012) and Maisetti (2012) [2, 5]. An attempt to estimate the resting muscle tension during passive motion of the ankle and knee joints was made mainly in the medial and lateral gastrocnemius and soleus muscles, where the slack angle was determined. The elastic modulus was increased by the muscle elongation, in which the muscle was stretched from the slack length [6–8].

Regarding the difference between elastic modulus and shear wave velocity, Eby (2013) found that the equation E = 3ρVs² (E: elastic modulus kPa, Vs: shear wave velocity m/s, ρ: muscle density 1000 kg/m³) holds if the density of the muscle is assumed to be uniform, in case of measurement along longitudinal muscle fiber direction [4]. On the contrary, if the density of muscles is not uniform, in case of measurement along transverse or oblique muscle fiber direction, it is expressed in terms of the anisotropic modulus (E = ρVs²) [2, 7]. In researches of the shear wave velocity in the leg muscles at a resting state (ankle position around the slack angle without muscle contraction), the muscle tissue was measured regarding as the isotropic modulus (E = 3ρVs²) [2, 5, 7–9].

The muscles of the lower leg are divided into four compartments: the anterior compartment consists of the tibialis anterior (TA) and the extensor hallucis longus and extensor digitorum longus, the anterior-lateral compartment consists of the peroneus longus and brevis, the superficial posterior compartment consists of the triceps surae muscle (the lateral and medial gastrocnemius (LG and MG), and soleus (SOL)), and the deep posterior compartment consists of the tibialis posterior and flexor hallucis longus and flexor digitorum longus. Compartment syndrome occurs when the internal pressure in each compartment increases due to excessive sports activity or severe trauma [10]. In addition, the gastrocnemius and soleus muscles in the posterior compartment contain large sinusoid veins, and venous thrombosis is known to occur when blood flow is stagnant [11].

There are several clinical conditions that cause lower leg symptom during a prolonged sitting, i.e. occupational leg edema after prolonged standing and sitting [12], thrombophlebitis of lower leg during long flight [13, 14] and fluid accumulation in the leg during long-distance bus travel [15]. To solve mechanism of prolonged sitting in lower leg, plethysmographic measurement for haemodynamics of calf veins or bioelectrical impedance analysis for tissue fluid has been performed [16, 17], but no measurement of resting leg muscles by shear wave elastography has been reported.

The purpose of this report is to measure the shear wave velocity of the tibialis anterior, gastrocnemius and soleus muscles by sitting for 2 hours and then raising the leg, and to clarify the physical changes of the leg muscles in the compartment.

We hypothesized that the shear wave velocity of the medial and lateral gastrocnemius, soleus and tibialis anterior muscle would increase with time in the resting leg position after 2 hours of sitting and decrease with subsequent leg elevation. Then we considered the mechanism of the velocity change with two points of view, i.e. one is change of leg muscle fibers and the other is change of extra-cellular space in the compartment.

Materials and methods

Subjects

Twenty-four healthy adult males (mean age, 26.6 ± 6.1 years) were included in the study. Subjects were recruited by announcements on posters which were exhibited in a student bulletin board. Subjects with blood disorders or traumatic diseases of the lower legs were excluded in advance. The subject’s height (172.4±7.4 cm), weight (65.8±7.8 kg), and BMI (22.0±2.5) were measured, and lower extremity muscle mass (9.8±1.0 kg), body fat mass (14.4±9.3 kg), and trunk muscle mass (26.0±2.5 kg) were measured using a Tanita body composition analyzer (MC-180, Tanita Ltd., Tokyo).

The purpose of the study, management of personal information, prohibition of using personal information for other purposes, and free participation in the study were explained to the subjects and their written consent was obtained. The individual in this manuscript has given written informed consent to publish obtained data including images of the participants. In accordance with the Declaration of Helsinki, the subject’s personal right and data protection was explained. This study was conducted with the approval of the University of Health Sciences Research Ethics Review Committee (approval number, 19R115109).

Experimental protocol

Subjects were seated in a reclining chair and held in a relaxed position for 2 hours. Shear wave velocity of the soleus, medial and lateral gastrocnemius, and tibialis anterior muscles of the left lower extremity were measured immediately after the start of a sitting, 60 minutes and 120 minutes later using an ultrasound system (Aplio 500 Canon, Tokyo). Afterwards, the patient raised left lower limb for 3 minutes and returned to the sitting position for measurement (Fig 1). Measurements were performed by a clinical laboratory technician (K.O.). The temperature and humidity in the laboratory were maintained at 25°C and 30–40%, respectively.

Shear wave velocity was measured after start of sitting on a reclining chair, at 60 min, and at 120 min. After 3 min leg raise on the stool, the velocity was measured again in sitting position.

Sitting posture

The subject was seated in a reclining chair and maintained 20 degrees of flexion at the ankle joint, 70 degrees of flexion at the knee joint, and 80 degrees of flexion at the hip joint as measured by a goniometer. In this limb position, the ankle is more flexed than the slack angle, which relaxes the triceps surae muscle [6–8]. In this limb position, the ankle joint was flexed for the tibialis anterior muscle to keep slightly elongated. The subjects were instructed to sit in a seated position with the width of the right and left legs shoulder-width apart and to maintain a relaxed state during the measurement, and the shear wave velocity was measured. The foot was held on the weight-bearing platform so that the foot load was equally applied to both soles (Fig 2A).

Fig 2 — a. Sitting position on a reclining chair. b. Leg raise on a stool.

Elevation of the lower limb

After 120 minutes of measurement, subjects were seated in a reclining chair with the knee joint extended and the lower leg raised on a stool at the same height as the thigh for 180 seconds to relax the lower leg and ankle, and then returned to the sitting position to measure the shear wave velocity (m/s) (Fig 2B). Participants remained in the same position sitting in a chair from the beginning until the end of the elastography measurement, except 3 minutes leg elevation.

Measurement of shear wave velocity

Shear wave velocity was measured as an indicator of muscle stiffness. We used an ultrasound system (Aplio 500, Canon, Tokyo) and a linear probe (PLT100BT5, Canon, Tokyo) with a field view of approximately 58 mm, standard operating frequency of 10 MHz (up to 14 MHz). The accuracy of the velocity measurement by Aplio500 using a phantom (CIRS, Norfork, Virginia, USA; model 049 and 049A) of 8.0 ± 3.0 kPa is 0.49 kPa, and the precision (Coefficient of Variation and Confidence Interval) is 6.96%, 5.79–8.13%. The accuracy and precision of the velocity measurements from 2 to 6 cm material depth are not different [18]. The range of measurement of the shear wave velocity of the leg muscles in this study by Aplio500 was 1.0–3.0 m/s (3.0–9.0 kPa), and the depth of the soleus muscle at the deepest point was about 4.0 cm, so the reliability of the measurement of the shear wave velocity of the leg muscles by Aplio 500 was assured.

The measured muscles were the medial and lateral gastrocnemius, soleus and tibialis anterior. The location of a probe to be applied on the muscle belly of the medial and lateral gastrocnemius, soleus and tibialis anterior muscles was adjusted in the designated position (the medial and lateral portions of the gastrocnemius muscle at 30% of the proximal left lower leg, the soleus muscle at 50% of the left lower leg, and the tibialis anterior muscle at 30% of the proximal left lower leg [2, 5]) for individual subject. On the most prominent part of the muscle belly, the rectangular shape skin mark with adhesive tape was attached prior to the measurement, and the probe was placed in the rectangular mark (Fig 3A and 3B). Since orientation of muscle fascicles of the gastrocnemius, soleus is oblique by their pennated structure and is redundant due to muscle relaxation, prove setting was aligned to anatomical muscle shortening direction.

Fig 3 — a. Sites of recording of shear wave velocity at lateral, medial gastrocnemius, and soleus muscles. b. Sites of recording of shear wave velocity at tibialis anterior muscle.

Ultrasound images were recorded along the long axis of each muscle. The probes were scanned parallel to the superficial fascia of each muscle. The size of the Region of Interest (ROI) was set to 10×10 mm². The area of analysis (Target ROI) was set as a circle of 5 mm in diameter in the center of the ROI [19]. The ultrasound images were taken three times for each muscle and the average of the three images obtained was calculated (Fig 4).

Fig 4 — LG: Lateral gastrocnemius, MG: Medial gastrocnemius, SOL: Soleus, TA: Tibialis anterior. ROI: Region of interest: 1 cm x 1 cm.

The shear wave velocity of the gastrocnemius and soleus muscles was measured at a value with the ankle joint in 20 degrees of flexion and muscle length was shorter than the slack length [20]. The shear wave velocity of the tibialis anterior muscle was measured with the ankle joint in 20 degrees of flexion at 10 degrees over the slack angle and with the muscle length slightly longer than the slack length [21]. Taking both longitudinal measurements of the muscle and muscle length into consideration, in the present measurements, we regarded both muscles as isotropic and elastic modulus was calculated by the following equation [7].

E = 3 ρ V s^{2}

Surface electromyography

In order to confirm the contraction of the leg muscles in the reclining seat, electromyography was measured by placing active wireless surface electrodes (Nihon Koden Ltd., Tokyo, Japan) with a distance of 10 mm between electrodes parallel to the muscle fibers in the medial and lateral gastrocnemius, soleus muscle and tibialis anterior muscle. Surface electrode placement sites were adjusted according to Hermens et al. (2000) [22]. The electromyography data was recorded at 2000 Hz and collected to a polygraph system of Web-1000 (Nihon Koden Ltd., Tokyo, Japan), then analyzed using LabChart version 7 (AD Instruments, Tokyo, Japan). During shear wave velocity measurement by ultra-sonography, EMG data was continuously displayed for examiner and subject on PC screen, suggesting the meaning of appearance of muscle activation waves for ensuring proper relaxation. Appearance of EMG activity was determined by observation of action potentials greater than 2SD amplitude of baseline. It was determined that no muscle contraction was observed to occur in our lower leg sitting position.

Reliability analysis

In order to evaluate the intra-observer reliability and accuracy of shear wave velocity measurements, shear wave velocity was measured in the medial and lateral gastrocnemius, soleus and tibialis anterior muscles in reclining seated sitting position in eight healthy male adults. Measurements were taken immediately after sitting and 20 minutes later, under the same conditions as in the present experiment, to obtain the intra-day intra-observer reliability (1, 3) and the coefficient of variation (CV). Intra-day intra-observer reliability and coefficient of variation (CV) for the lateral gastrocnemius muscle was 0.88 and 3.9%, for the medial gastrocnemius, 0.87 and 5.7%, for the soleus muscle, 0.92 and 7.5%, and for the tibialis anterior muscle, 0.89 and 14.2%. After one more day, measurements were performed again, the inter-day intra-observer reliability (1, 3) and the coefficient of variation (CV) was obtained. The inter-day intra-observer reliability and the correlation coefficient (CV) for the lateral gastrocnemius was 0.77 and 3.7%, the medial gastrocnemius was 0.82 and 6.5%, the soleus was 0.76 and 8.7%, and the tibialis anterior was 0.81 and 12.0%.

Sample size

To determine the sample size for repeated measures two-way analysis of variance, we used Cohen’s moderate effect size of 0.25, calculated the group as 2, the level as 4, the α value as 0.05 and the β value as 0.8, and set the sample size as 24 [23]. Therefore, the number of subjects in this study was set as 24.

Statistical analysis

Information of subjects were analyzed by the Shapiro-Wilk test, and the normality of all values was confirmed. The measured shear wave velocities from four different muscles was analyzed by the Shapiro-Wilk test, and the normality of all the values was confirmed. Measurements obtained with time were analyzed by repeated-measures two-way analysis of variance, and no interaction was found for the combination of LG and TA or MG and TA. However, because there were interactions between SOL and TA, and between LG, MG, and SOL with each other, repeated-measures one-way analysis of variance was performed for individual muscles. The Bonferroni method was used for the post-hoc test. A corresponding t-test was used for inter-muscle comparisons of shear wave velocities of immediately after the start of a sitting. Statistical analysis was performed using SPSS ver23, and level of significance was set at 0.05.

Result

Change in the shear wave velocity of the leg muscles with time

In the lateral and medial gastrocnemius muscles, the shear wave velocity increased significantly at 60 minutes (1.58 ± 0.06, 1.70 ± 0.09 m/s) and 120 minutes (1.70 ± 0.10, 1.83 ± 0.11 m/s) compared to immediately after the start of sitting, respectively (1.52 ± 0.06, 1.66 ± 0.10 m/s) (p<0.01, p<0.05). And the velocity after leg raising (1.52 ± 0.07, 1.59 ± 0.07 m/s) is significantly lower than the velocity at 120 minutes, respectively (p<0.01, p<0.01) (Table 1, Figs 5 and 6).

Table 1. Shear wave velocity of LG, MG, SOL, and TA over time.

	Start of sitting	60 min	120 min	After leg raise
LG	1.52 ± 0.06	1.58 ± 0.06^*	1.70 ± 0.10^*	1.52 ± 0.07^**
MG	1.66 ± 0.10	1.70 ± 0.09^*	1.83 ± 0.11^*	1.59 ± 0.07^*
SOL	1.60 ± 0.15	1.69 ± 0.15	1.89 ± 0.17^*	1.55 ± 0.11^**
TA	2.15 ± 0.26	2.21 ± 0.24	2.30 ± 0.24^*	2.03 ± 0.22^**

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Unit: m/s.

Average ± SD.

LG: Lateral gastrocnemius, MG: Medial gastrocnemius, SOL: Soleus, TA: Tibialis anterior

*p<0.05, Shear wave velocity is greater than that of start of sitting.

**p<0.01, Shear wave velocity of after leg raise is smaller than that of 120 min.

In the soleus and tibialis anterior muscles, the shear wave velocity increased significantly after 120 minutes (1.89 ± 0.17, 2.30 ± 0.24 m/s) compared to immediately after the start of sitting (1.60 ± 0.15, 2.15 ± 0.26 m/s), respectively (p<0.01, p<0.05). And the velocity after leg raising (1.55 ± 0.11, 2.03 ± 0.22 m/s) is significantly lower than the velocity at 120 minutes, respectively (p<0.01, p<0.01). (Table 1, Figs 7 and 8).

Comparison of shear wave velocity between muscles immediately after the start of sitting

The shear wave velocity of the tibialis anterior muscle was significantly greater than that of the other muscles (p<0.01) (Table 1). There was no difference in the shear wave velocity among the medial and lateral gastrocnemius and soleus muscles, except the shear wave velocity between the lateral gastrocnemius and medial gastrocnemius muscle (p<0.01).

Discussion

Taniguchi (2015) reported that in the lateral gastrocnemius and medial gastrocnemius muscles stretched beyond the slack length, the elastic modulus was unchanged (7.5 kPa in the lateral gastrocnemius and 10.5 kPa in the medial gastrocnemius) after 20 minutes of resting in the standing position [6]. There is no other report on the measurement of the shear wave velocity in the leg muscles under the prolonged resting condition.

In this study, we observed the shear wave velocity of the leg muscles in sitting position and its change with time. As the gastrocnemius and soleus muscle located behind the lower leg were measured in a relaxed position, their length is shorter than the slack length. As the tibialis anterior muscle located anterior to the lower leg was measured at the ankle flexed position, its length is longer than the slack length. Shear wave velocity in the lateral gastrocnemius, medial gastrocnemius, and soleus muscles increased significantly during 120 minutes of sitting compared to the immediately after sitting and decreased significantly during 3 min leg raising (Table 1, Figs 5–7). The shear wave velocity in the tibialis anterior muscle was significantly greater than those in the gastrocnemius and soleus muscles (p<0.01). It increased significantly with time, and decreased significantly after elevation of the lower leg (Table 1, Fig 8).

Toyoshima (2020) reported using Turkey legs that the shear wave velocity increased proportionally with the increase in pressure of the tibialis anterior compartment [24]. Specifically, when the internal pressure of the tibialis anterior muscle is 1–2 mmHg, the shear wave velocity (elastic modulus) is 2.5 m/s (6.25 kPa) and increases linearly from 3.0 m/s (9.0 kPa) to 3.5 m/s (12.5 kPa) as the internal pressure increases from 10 mmHg to 20 mmHg. In the reports of human measurement, shear wave velocity (elastic modulus) of the tibialis anterior muscle at rest has been reported to be 2.1–3.2 m/s (4.4–10.2 kPa) [4, 9, 22, 23]. In our measurements, the shear wave velocity (elastic modulus) of the tibialis anterior muscle was 2.17 m/s (6.51 kPa), which is similar to the previous reports (Table 1, Fig 8).

In sitting position, venous pressure of popliteal vein at the popliteal fossa and pressure of saphenous vein at the medial malleolus were reported to be 23.9 mmHg (3.17 kPa) and 54.2 mmHg (7.2 kPa), respectively [25]. Considering the location of shear wave velocity measurements in the tibialis anterior and gastrocnemius muscles in the sitting position, it can be inferred that hydrostatic pressure is equally applied to the tibialis anterior and gastrocnemius muscles through the leg veins for two hours.

In the present measurement, the shear wave velocity of the tibialis anterior muscle was significantly greater than that of the gastrocnemius and soleus muscles at any times (Table 1). This is because the tibialis anterior muscle is stretched about 10% longer than the slack length at the sitting leg position, which may be one of the reasons why the shear wave velocity is higher than that of the relaxed gastrocnemius muscle [6]. The lateral gastrocnemius muscle had a smaller shear wave velocity than the medial gastrocnemius muscle, but this is unlikely to be due to a difference in muscle length, and is assumed to be due to the pennation angle of muscle fibers or muscle fiber length. The similar difference in shear modulus was observed in healthy volunteers between the lateral and medial gastrocnemius muscle [6].

Increase of muscle stiffness in active muscle conditions is reported to have several causes, i.e. reflex-mediated stiffness by prolonged muscle contraction after repetitive task which lead to the release of metabolites and activate nociceptors [26, 27], temporary increase of muscle stiffness after repetitive muscle contraction due to static or dynamic exercises, which is reflected by increased intramuscular pressure associated with increases in intramuscular blood flow [28, 29], or static stretch of the muscles by which muscle stiffness increase proportionally to the muscle length [6]. Increase of muscle stiffness in resting muscle conditions is reported to have causes, i.e. increasing collagen mass in muscles by aging and spastic conditions with neuromuscular diseases [7, 30].

On the other hand, although no measurement by shear wave elastography has been performed, the another suspected causes of increase in muscle stiffness in resting muscles are reported to be fluid retention in the leg due to occupational leg edema after prolonged standing and sitting [12], long-haul flight simulation of sitting 4 to 12 hours [16], and the effect of 4 ours sitting and calf activity [17]. Belczak et al (2018) reported significant increase in leg volume by volumetric measurement using water displacement technique [12]. Mittermayr et al (2007) reported increased lower leg volume by plethysmographic measurement using an optoelectronic scanner system, suggesting the phenomena was due to extravascular fluid shifts in the lower leg. They also measured by ultrasonography the cross-sectional diameter of calf veins, there was no significant change in the diameter by 4 to 12 hours sitting [16]. Singh et al (2017) reported increased leg volume estimated by calf circumference and by bioelectrical impedance analysis. In their study, they suggested two points, first increase in immediate intravascular leg fluid due to hydrostatic pressure and a loss of leg muscle tone, second retention of extravascular fluid in the legs by hydrostatic pressure after 4 hours sitting [17].

Based on literature and result of our study, increase in shear wave velocity with prolonged sitting does not come from change of leg muscle fibers or connective tissue but may come from increase in intra-compartment pressure of the lower leg due to fluid retention in extra-cellular space.

It is presumed that the shear wave velocity decreased by leg elevation without delay because of the drainage of intra-venous blood out of lower leg, resulting in a decrease in intramuscular pressure (Table 1) [11]. This means that the blood in the muscle compartment is drained out. Even if the venous valves work in the lower leg in healthy adults, gradual leaking of the blood exudate might occur, resulting in increase in intramuscular pressure with time. However, at this point, we have not measured intramuscular pressure or venous blood flow, therefore it is only possible to estimate the change in internal pressure in the gastrocnemius and tibialis anterior muscle by changes in shear wave velocity.

During a long sitting period in a recliner, the knee joint is often kept at nearly 90 degrees of flexion and the ankle joint is kept in slight flexion. The results of this study have clinical importance in the possibility of prevention of muscle stiffness and edema of the lower legs during long time flight or evacuation in the car as refugees after huge earth quake. Measurement of shear wave velocity by non-invasive ultrasonography help people know the extent of leg edema in timely fashion [14, 31]. In such cases, it is important to know that simple elevation of lower leg for 3 minutes may reduce leg edema.

Research limitations

Measurement of muscle at rest

In our study, we measured the leg immobile in the sitting position for a long time. However, except in special cases, it is rare to have a situation in which the subjects do not move his or her legs for 2 hours during sitting. In the future, it is necessary to make ultrasound measurements that take the motion of the lower limbs into account.

Measurement for adult male

In this study, only adult males were included in the measurements, but venous blood retention originally occurs more frequently in middle aged women and is observed as varicose veins and leg edema [11]. In the future, with the approval of the ethics committee, we will add measurements on the lower legs of aged women.

Measurement up to 2 hours

In this study, we adopted the lower leg sitting condition for up to 2 hours. According to previous reports, the occurrence of economy class syndrome is known to occur after a flight longer than 6 hours. While avoiding the development of venous thrombosis, measurements should also be performed under conditions exceeding 2 hours [14, 31].

No intramuscular pressure measurement is performed.

In this measurement, the internal pressure of the leg muscle compartment was estimated as the change in the value of the shear wave velocity. For more direct measurement, intramuscular pressure measurement is required [4, 25].

Venous hemodynamics

In the present measurement, blood retention in the vein was not assessed. Since ultrasonic measuring devices can measure the increase in the diameter of the intramuscular veins and blood flow, there is a need to investigate the hemodynamics of venous blood in conjunction with the measurement of shear wave velocity [11, 24].

Conclusions

Shear wave velocity of the lower leg muscles increased with time in 2 hours sitting, and decreased with subsequent leg elevation. Based on the report that the change in the shear wave velocity was proportional to the internal pressure of the leg muscle compartment in turkey models, it is estimated that increase in shear wave velocity with prolonged sitting may come from increase in intra-compartment pressure of the lower leg due to fluid retention in extra-cellular space of the compartment.

Supporting information

S1 Table. Shear wave velocity of the lower leg muscles (n = 24).

(DOCX)

Click here for additional data file.^{(20.1KB, docx)}

Acknowledgments

We express special thanks to all contributors (Tomoya Hayashi, Hisashi Honma, Yuji Sasaki, Kazuyuki Sugawara) for assistance of experiments.

Data Availability

All relevant data are within the paper and its Supporting Information file.

Funding Statement

The authors received no specific funding for this work.

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PLoS One. doi: 10.1371/journal.pone.0251532.r001

Decision Letter 0

Guy Cloutier

9 Feb 2021

PONE-D-20-36699

Effect of prolonged sitting immobility on shear wave velocity of the lower leg muscles

PLOS ONE

Dear Dr. Aoki,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Although mainly descriptive your results might be of value to the community.

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Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Partly

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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Reviewer #1: General comments :

This study assessed shear wave velocity of lower limb muscles (gastrocnemius, soleus and tibialis anterior muscles) at baseline, after prolonged sitting (60 min and 120 min) and in a leg-raised position. The study seems well-conducted with a sufficient sample size and an adequate methodology. However, the manuscript would benefit being reviewed by a professional proofreader/editor to improve the language.

Specific comments:

P4-5: Please describe the inclusion and exclusion criteria. The information about where and how the participants were recruited should be provided. The characteristics of the participants (Table 1) should be moved to the result section. The related statistics and the test used to assess the normality of the data should be detailed in the statistical section of the methodology.

P5, L122: the number of participants should be in the participant section of the methodology or the results section.

P5, L128: the reference to the Figure 1 is not related to the text “The temperature and humidity in the laboratory were maintained at 25°C and 30-40%, respectively”.

P7, L167-168: Positioning of the probe on the gastrocnemius, soleus and tibialis anterior muscles. Which landmarks were used to establish the positioning of the probe (e.g. 30% and 50% of the leg). How was the muscle belly located?

P7: Did the participants remain in the same position for the elastography measurements? It is not clear that the surface scanned was easily accessible for the evaluator according the image provided.

P8: Surface electromyography. Can you further describe how the EMG data were managed during the entire testing period? Were the EMG traces visible for both the participants and the evaluator? Was a pre-established resting activity targeted on a screen for ensuring proper relaxation?

P9, L235: Replace “the risk rate was set at” by “level of significance was set at”

P 11 L283- 297: In the discussion, the values obtained should not be repeated. The results should rather be summarized and discussed according the current literature.

P12, L33-335: Do you have any reference supporting this hypothesis? The authors should explain the hypothesized mechanisms responsible for the increase in shear wave velocity can with prolonged sitting and they should support their explanations with the literature.

Reviewer #2: The reviewer has thoroughly reviewed the manuscript for scientific content, manuscript construction, and general impact. This study seems a sound one but needs to be redefined, especially in terms of novelty, hypothesis, and methodology. The main issues are listed below.

Introduction

The introduction section should be clear and hypothesis-driven to allow the readers to see the basis of the authors’ hypothesis. Although the hypothesis is stated in the manuscript, the reviewer cannot understand the rationale for the hypothesis. Additionally, in the introduction section, the authors should not exhaustively review the subject but need to provide relevant references. Namely, the authors should mention the literature on muscle conditions during a prolonged sitting.

The authors consider that the clinical importance of the present study is related to compartment syndrome, it would be better to measure intramuscular pressure instead of shear wave velocity.

Methods

The authors report 3ρVs2 because they regarded the muscles as isotropic (Line 190-). However, this is wrong (although the authors are aware).

The orientation of the ultrasound probe is unclear. As far as the reviewer see Figure 4, it seems that the probe was not aligned with the fascicle direction of the muscles. As the authors know, the probe orientation relative to fascicle direction affects the shear wave speed measured, leading to smaller shear wave velocity. In fact, the shear wave velocity reported in this paper is smaller than those in previous studies.

The ICC in this study is smaller than that of previous studies (ICC > 0.9).

The authors stated that no muscle contraction was observed to occur through EMG recording. Please explain in more detail what value the authors determined to be no muscle contractions.

Discussion

The discussion section needs to reflect what the authors found, how it relates to the literature, and then what it means clinically or physiologically. Each paragraph should be logical in sequence as at present; it is a bit hard to follow. Further, the discussion is qualitative but not quantitative.

Conclusion

Intramuscular pressure was not directly measured in this paper. Thus, the current conclusion is not based on the data of the present study.

Reviewer #3: PONE-D-20-36699

This study brings descriptive data to the literature on the effect of prolonged sitting on lower limb muscle shear wave velocity (i.e., muscle stiffness under known hypotheses of localized homogeneous, isotropic and purely linear elastic property, which oversimplify the muscle rheology but is of common use in the field). If the angle of insonification with respect to the muscle long axis is defined, this can bring informative data to the literature. The study protocol is quite simple and the sample size is limited in term of number and population characteristics; only normal young male volunteers were scanned. EMG verification of the absence of muscle contraction, which is an important confounder, was made to improve robustness of reported results. Overall, even though it is mainly a descriptive study with no clear rationale or hypothesis, reported results might be of value to design future studies on pathological cohorts of patients with musculoskeletal disorders. A strong effort in restructuring the paper is required, many sections are not at the right place into the manuscript and repetitions are noticed. Please use standard scientific report guidelines.

1) By considering the limited sample size, I would add “a proof-of-concept study” at the end of the title.

2) Abstract, L38-39: There is repetitive information on statistics.

3) Introduction, L76: The concept of anisotropic modulus is confusing. L73 is giving the Young’s modulus estimate for a linear, isotropic and purely elastic material, whereas the equation of L76 is simply the shear modulus under same hypotheses. There is no anisotropic modulus leading to this simplified equation, as far as I know.

4) L87: The concept of compartmental pressure increase is indeed of interest to interpret the dataset as the boundary condition imposed by the internal pressure on muscles changes the non-linear strain-stress relation of most biological tissues. Any pre-compressed tissue will result in a displacement of the measured elasticity on the non-linear strain-stress curve and thus will lead to a different estimate of the Young’s modulus given by the equation on L73. This is a limitation of shear wave elastography imaging as linear elasticity is assumed. It would be important to bring basic tissue mechanics concepts into your discussion to avoid misleading interpretation.

5) L97: Your hypothesis is empty and not based on strong rationales. This should be improved. Why prolonged sitting would change muscle stiffness?

6) Table 1 is useless and should be removed.

7) L159: “precision”.

8) L186 and 188: What is a “shear wave verbosity” and what is “a constant condition”?

9) L203: What is the output of the Web-1000 system?

10) Discussion: Remove all repetitions on reported results, this is useless.

11) L309 and elsewhere: Correct “verbosity”.

12) L315: Are you sure about this curious hypothesis? How the hydrostatic pressure applied on muscles can be induced by the venous low pressure system of typically a few mmHg? This sounds curious to me.

13) L317: You are overstretching the interpretation of your results since you did not measure the intramuscular pressure; consequently you cannot say that this is “in accordance with”.

14) L327: The muscle length has certainly nothing to do with shear wave speed; you are likely referring to the muscle stretching.

15) L328: “nature of muscle”, can you be more descriptive and support your hypothesis based on known histopathology analysis?

16) L330: Here again, verify your hypothesis about venous pressure and impact on muscle. Swelling of muscle versus direct impact of venous pressure are likely different mechanisms. L332: Even though you are citing the literature, more rationale should be given on how an intravenous pressure of typical < 10 mmHg would compress the muscle enough to change the strain-stress non-linear elasticity behavior.

17) L342: Indeed assessing the venous system with Doppler flow and B-mode diameter measurements would have helped supporting your hypothesis; otherwise your study is mainly descriptive. An impedance plethysmography system would also help assessing electrolyte volume (i.e., blood volume and interstitial inter-compartment water).

18) Conclusion: Here again you are overstretching the interpretation of your results; no internal pressure has been reported.

19) L391: “evacuation in the car”, what do you mean?

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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PLoS One. 2021 May 10;16(5):e0251532. doi: 10.1371/journal.pone.0251532.r002

Author response to Decision Letter 0

17 Mar 2021

Reply to the Journal requirement:

The title of the manuscript was revised in all uploaded documents, including cover letter, manuscript, and rebuttal letter as follows: “Effect of prolonged sitting immobility on shear wave velocity of the lower leg muscles in healthy adults: a proof-of-concept study”

Line117, (Line119-124): All ethics statements of participants were moved to the Method section.

Line119, (Line121-122): Statements of consent for publication of the individual who appeared in the text was provided as follows: “The individual in this manuscript has given written informed consent to publish these case details.”

Following marks, Line**-**, (Line**- **), suggests Line on the original manuscript, and (Line on the revised manuscript).

Reply to Reviewer #1, #2, #3

Reviewer #1: General comments:

Specific comments:

Line105, (Line112-113): Information about recruitment of participant was stated in the Method section as follows: “Subjects were recruited by announcements on posters which were exhibited in a student bulletin board.”

Table 1 was removed.

Line110, (Line236-237): Normality of the data was moved to statistics section.

P5, L122: the number of participants should be in the participant section of the methodology or the results section.

Line122: Number of participants was removed.

P5, L128: the reference to the Figure 1 is not related to the text “The temperature and humidity in the laboratory were maintained at 25°C and 30-40%, respectively”.

Line128, (Line131): Reference of (Figure.1) was moved up to adequate location.

Line 167-171, (Line169-178): The prove positioning and location on the muscle belly was stated specifically as follows: “The location of a probe to be applied on the muscle belly of the medial and lateral gastrocnemius, soleus and tibialis anterior muscles was adjusted in the designated position (the medial and lateral portions of the gastrocnemius muscle at 30 % of the proximal left lower leg, the soleus muscle at 50 % of the left lower leg, and the tibialis anterior muscle at 30 % of the proximal left lower leg [2, 5]) for individual subject. On the most prominent part of the muscle belly, the rectangular shape skin mark with adhesive tape was attached prior to the measurement, and the probe was placed in the rectangular mark (Fig 3a, 3b). Since orientation of muscle fascicles of the gastrocnemius, soleus is oblique by their pennated structure and is redundant due to muscle relaxation, prove setting was aligned to anatomical muscle shortening direction.”

P7: Did the participants remain in the same position for the elastography measurements? It is not clear that the surface scanned was easily accessible for the evaluator according the image provided.

Line 142, (Line154-155): “Participants remained in the same position sitting in a chair from the beginning until the end of the elastography measurement, except 3 minutes leg elevation.” This statement was added at the end of “Elevation of the lower limb”.

Line168-171, (Line169-178): A prove positioning and location of the muscle belly was stated specifically as follows: “The location of a probe to be applied on the muscle belly of the medial and lateral gastrocnemius, soleus and tibialis anterior muscles was adjusted in the designated position (the medial and lateral portions of the gastrocnemius muscle at 30 % of the proximal left lower leg, the soleus muscle at 50 % of the left lower leg, and the tibialis anterior muscle at 30 % of the proximal left lower leg [2, 5]) for individual subject. On the most prominent part of the muscle belly, the rectangular shape skin mark with adhesive tape was attached prior to the measurement, and the probe was placed in the rectangular mark (Fig 3a, 3b). Since orientation of muscle fascicles of the gastrocnemius, soleus is oblique by their pennated structure and is redundant due to muscle relaxation, prove setting was aligned to anatomical muscle shortening direction.”

Line203, (Line208-212): Statement of traceability of EMG data during elastography measurement for the evaluator was added as follows: “During shear wave velocity measurement by ultra-sonography, EMG data was continuously displayed for examiner and subject on PC screen, suggesting the meaning of appearance of muscle activation waves for ensuring proper relaxation.”

P9, L235: Replace “the risk rate was set at” by “level of significance was set at”

Line235, (Line245): “the risk rate was set at” was replaced by “level of significance was set at” as suggested.

P 11 L283- 297: In the discussion, the values obtained should not be repeated. The results should rather be summarized and discussed according the current literature.

L283- 297, (Line287-373): All repetitions on reported results were removed from the Discussion

P12, L333-335: Do you have any reference supporting this hypothesis? The authors should explain the hypothesized mechanisms responsible for the increase in shear wave velocity can with prolonged sitting and they should support their explanations with the literature.

Line97, (Line102-106): The hypothesis was added by providing specific pathology in the lower leg muscles.

Line333-335, (Line331-358): The hypothesized mechanism was provided in two points of view, i.e. one is change of leg muscle fibers and the other is change of extra-cellular space in the compartment.

Introduction

The authors consider that the clinical importance of the present study is related to compartment syndrome, it would be better to measure intramuscular pressure instead of shear wave velocity.

Line97, (Line102-106): The hypothesis was added by providing specific pathology in the lower leg muscles as follows: “We hypothesized that the shear wave velocity of the medial and lateral gastrocnemius, soleus and tibialis anterior muscle would increase with time in the resting leg position after 2 hours of sitting and decrease with subsequent leg elevation. Then we considered the mechanism of the velocity change with two points of view, i.e. one is change of leg muscle fibers and the other is change of extra-cellular space in the compartment.”

Line91, (Line91-96): In the middle of Introduction, estimated muscle condition during a prolonged sitting was listed by mentioning literature as follows: “There are several clinical conditions that cause lower leg symptom during a prolonged sitting, i.e. occupational leg edema after prolonged standing and sitting [12], thrombophlebitis of lower leg during long flight [13, 14] and fluid accumulation in the leg during long-distance bus travel [15]. To solve mechanism of prolonged sitting in lower leg, plethysmographic measurement for haemodynamics of calf veins or bioelectrical impedance analysis for tissue fluid has been performed [16, 17], but no measurement of resting leg muscles by shear wave elastography has been reported.”

”.

Line329-, (Line331-339): In Discussion, to verify hypothesis about venous pressure and impact on muscle, speculated pathology occurred in the lower leg muscles was stated with providing literature as follows: “Increase of muscle stiffness in active muscle conditions is reported to have several causes, i.e. reflex-mediated stiffness by prolonged muscle contraction after repetitive task which lead to the release of metabolites and activate nociceptors [26, 27], temporary increase of muscle stiffness after repetitive muscle contraction due to static or dynamic exercises, which is reflected by increased intramuscular pressure associated with increases in intramuscular blood flow [28, 29], or static stretch of the muscles by which muscle stiffness increase proportionally to the muscle length [6]. Increase of muscle stiffness in resting muscle conditions is reported to have causes, i.e. increasing collagen mass in muscles by aging and spastic conditions with neuromuscular diseases [7, 30].”

“On the other hand, although no measurement by shear wave elastography has been performed, the another suspected causes of increase in muscle stiffness in resting muscles are reported to be fluid retention in the leg due to occupational leg edema after prolonged standing and sitting [12], long-haul flight simulation of sitting 4 to 12 hours [16], and the effect of 4 ours sitting and calf activity [17]. Belczak et al (2018) reported significant increase in leg volume by volumetric measurement using water displacement technique [12]. Mittermayr et al (2007) reported increased lower leg volume by plethysmographic measurement using an optoelectronic scanner system, suggesting the phenomena was due to extravascular fluid shifts in the lower leg. They also measured by ultrasonography the cross-sectional diameter of calf veins, there was no significant change in the diameter by 4 to 12 hours sitting [16]. Singh et al (2017) reported increased leg volume estimated by calf circumference and by bioelectrical impedance analysis. In their study, they suggested two points, first increase in immediate intravascular leg fluid due to hydrostatic pressure and a loss of leg muscle tone, second retention of extravascular fluid in the legs by hydrostatic pressure after 4 hours sitting [17].”

Line344, (Line356-358): The following summarized statements was inserted.

“Based on literature and result of our study, increase in shear wave velocity with prolonged sitting does not come from change of leg muscle fibers or connective tissue but may come from increase in intra-compartment pressure of the lower leg due to fluid retention in extra-cellular space.”

Line347, (Line370-373): Clinical importance was stated about detection and treatment of leg edema with measurement of shear wave velocity by non-invasive ultrasonography as follows: “The results of this study have clinical importance in the possibility of prevention of muscle stiffness and edema of the lower legs during long time flight or evacuation in the car as refugees after huge earth quake. Measurement of shear wave velocity by non-invasive ultrasonography help people know the extent of leg edema in timely fashion.”

Methods

The authors report 3ρVs2 because they regarded the muscles as isotropic (Line 190-). However, this is wrong (although the authors are aware).

The ICC in this study is smaller than that of previous studies (ICC > 0.9).

The authors stated that no muscle contraction was observed to occur through EMG recording. Please explain in more detail what value the authors determined to be no muscle contractions.

Line167-171, (Line169-178): Detail placement of probe and location on the muscle belly was stated in the text as follows: “The location of a probe to be applied on the muscle belly of the medial and lateral gastrocnemius, soleus and tibialis anterior muscles was adjusted in the designated position (the medial and lateral portions of the gastrocnemius muscle at 30 % of the proximal left lower leg, the soleus muscle at 50 % of the left lower leg, and the tibialis anterior muscle at 30 % of the proximal left lower leg [2, 5]) for individual subject. On the most prominent part of the muscle belly, the rectangular shape skin mark with adhesive tape was attached prior to the measurement, and the probe was placed in the rectangular mark (Fig 3a, 3b). Since orientation of muscle fascicles of the gastrocnemius, soleus is oblique by their pennated structure and is redundant due to muscle relaxation, prove setting was aligned to anatomical muscle shortening direction.”

Line203-204, (Line211-212): Statement of EMG activity recording was corrected as follows: “Appearance of EMG activity was determined by observation of action potentials greater than 2SD amplitude of baseline.”

Discussion

Line329-, (Line331-358): We added in Discussion hypothesized mechanism with two points of view, i.e. one is change of leg muscle fibers and the other is change of extra-cellular space in the compartment.

Conclusion

Intramuscular pressure was not directly measured in this paper. Thus, the current conclusion is not based on the data of the present study.

Line384-391, (Line403-407): Conclusion was revised based on the result of increase in shear wave velocity and speculated pathology in the lower leg muscle as follows: “Shear wave velocity of the lower leg muscles increased with time in 2 hours sitting, and decreased with subsequent leg elevation. It has been reported that the change in the shear wave velocity was proportional to the internal pressure of the leg muscle compartment. Increase in shear wave velocity with prolonged sitting may come from increase in intra-compartment pressure of the lower leg due to fluid retention in extra-cellular space of the compartment.”

Line43-44, (Line45): Conclusion in Abstract was revised based on Conclusion in the Text as follows: “and it is assumed that the increase of shear wave velocity in the 2-hour seated leg is due to fluid retention in extra-cellular space of the compartment.”

Reviewer #3: PONE-D-20-36699

1) By considering the limited sample size, I would add “a proof-of-concept study” at the end of the title.

Line1, (Line1): “a proof-of-concept study” was added in the title as suggested.

2) Abstract, L38-39: There is repetitive information on statistics.

Line30-39, (Line39-40): The repetitive part was removed and data in parenthesis was corrected.

Line75-76, (Line75-79): The sentence “On the contrary, if the density of muscles is not uniform, it is expressed in terms of the anisotropic modulus (anisotropic E= ρVs2) [2,7].” was removed to avoid confusion. And the paragraph was revised as follows, “Regarding the difference between elastic modulus and shear wave velocity, Eby (2013) found that the equation E=3ρVs2 (E: elastic modulus kPa, Vs: shear wave velocity m/s, ρ: muscle density 1000 kg/m3) holds if the density of the muscle is assumed to be uniform, in case of measurement along longitudinal muscle fiber direction [4]. On the contrary, if the density of muscles is not uniform, in case of measurement along transverse or oblique muscle fiber direction, it is expressed in terms of the anisotropic modulus (E=ρVs2) [2,7]. In researches of the shear wave velocity in the leg muscles at a resting state (ankle position around the slack angle without muscle contraction), the muscle tissue was measured regarding as the isotrophic modulus (E= 3ρVs2) [2,5,7-9].”

5) L97: Your hypothesis is empty and not based on strong rationales. This should be improved. Why prolonged sitting would change muscle stiffness?

6) Table 1 is useless and should be removed.

Table 1 was removed and detail statement of this result was provided in the Results.

7) L159: “precision”.

Line159: typo error was corrected

8) L186 and 188: What is a “shear wave verbosity” and what is “a constant condition”?

Line186: “a constant” was removed as suggested.

9) L203: What is the output of the Web-1000 system?

Line203, (Line206-208): The sentence was corrected as follows: “The electromyography data was recorded at 2000 Hz and collected to a polygraph system of Web-1000 (Nihon Koden Ltd., Tokyo, Japan), then analyzed using LabChart version 7 (AD Instruments, Tokyo, Japan).”

10) Discussion: Remove all repetitions on reported results, this is useless.

L283- 297, (Line287-373): All repetitions on reported results were removed from the Discussion

11) L309 and elsewhere: Correct “verbosity”.

Line309: All typo error was corrected

Line309-320, (Line314-319): These two paragraphs were revised to state the effect of hydrostatic pressure in the low leg with sitting position to avoid confusions as follows: “In sitting position, venous pressure of popliteal vein at the popliteal fossa and pressure of saphenous vein at the medial malleolus were reported to be 23.9 mmHg (3.17 kPa) and 54.2 mmHg (7.2 kPa), respectively [25]. Considering the location of shear wave velocity measurements in the tibialis anterior and gastrocnemius muscles in the sitting position, it can be inferred that hydrostatic pressure is equally applied to the tibialis anterior and gastrocnemius muscles through the leg veins for two hours.”

13) L317: You are overstretching the interpretation of your results since you did not measure the intramuscular pressure; consequently you cannot say that this is “in accordance with”.

Line317: This paragraph was removed.

14) L327: The muscle length has certainly nothing to do with shear wave speed; you are likely referring to the muscle stretching.

Line327, (Line326): We consider the statement of muscle length in this context was correct.

15) L328: “nature of muscle”, can you be more descriptive and support your hypothesis based on known histopathology analysis?

Line328, (Line327): The phrase “nature of muscle” was replaced by “pennation angle of muscle fibers or muscle fiber length”

Line329-, (Line331-358): To verify hypothesis about venous pressure and impact on muscle, speculated pathology occurred in the lower leg muscles was stated with providing literature as follows: “Increase of muscle stiffness in active muscle conditions is reported to have several causes, i.e. reflex-mediated stiffness by prolonged muscle contraction after repetitive task which lead to the release of metabolites and activate nociceptors [26, 27], temporary increase of muscle stiffness after repetitive muscle contraction due to static or dynamic exercises, which is reflected by increased intramuscular pressure associated with increases in intramuscular blood flow [28, 29], or static stretch of the muscles by which muscle stiffness increase proportionally to the muscle length [6]. Increase of muscle stiffness in resting muscle conditions is reported to have causes, i.e. increasing collagen mass in muscles by aging and spastic conditions with neuromuscular diseases [7, 30].”

Line344, (Line356-358): The following summarized statements was inserted.

”

Line342, (Line341-354): Providing literature, statement of plethysmographic measurement and bioelectrical impedance analysis for leg edema was added in Discussion.

18) Conclusion: Here again you are overstretching the interpretation of your results; no internal pressure has been reported.

19) L391: “evacuation in the car”, what do you mean?

Line391, (Line370-373): We added statement of evacuation in the car as refugees after huge earth-quake as follows: “The results of this study have clinical importance in the possibility of prevention of muscle stiffness and edema of the lower legs during long time flight or evacuation in the car as refugees after huge earth quake. Measurement of shear wave velocity by non-invasive ultrasonography help people know the extent of leg edema in timely fashion [14, 33]”

At the time of big earthquake named “The east Japan big earthquake in 2011”, many refugees stayed in their own cars for weeks because of lack in housings. Significant number of evacuated people suffered from deep vein thrombosis.

Attachment

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Click here for additional data file.^{(38.5KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0251532.r003

Decision Letter 1

Guy Cloutier

9 Apr 2021

PONE-D-20-36699R1

Effect of prolonged sitting immobility on shear wave velocity of the lower leg muscles in healthy adults: a proof-of-concept study

PLOS ONE

Dear Dr. Aoki,

==============================

ACADEMIC EDITOR: I also reviewed your responses and I agree with Reviewer #3, a new round of revision with clear responses to all comments and associated changes into the manuscript should be provided. Take all advises given by this reviewer for not further delaying the decision.

==============================

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Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #3: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #3: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #3: No

**********

6. Review Comments to the Author

Reviewer #3: Overall, your responses to reviewers were badly formulated and it was very difficult to identify if all comments were properly answered. Each reviewer comment should be followed by a clear section indicating your response (e.g., RESPONSE TO COMMENT #1, RESPONSE TO COMMENT #2, etc ...). Also a different color code should be used for each reviewer to allow facilitating identifying changes made in the text. I could notice again the overstretching of the conclusion: "It has been reported that the change in the shear wave velocity was proportional to the internal pressure of the leg muscle compartment", you did not measure any internal pressure! Some typos were also noticed into changes made in the revised document.

No decision can be made unless a new "responses to reviewers" document is made with clear color-coded changes.

**********

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Reviewer #3: No

PLoS One. 2021 May 10;16(5):e0251532. doi: 10.1371/journal.pone.0251532.r004

Author response to Decision Letter 1

19 Apr 2021

PONE-D-20-36699R1

Reply to the comments of Academic Editor in the first revision

Line117, (Line121-125): All ethics statements of participants were moved to the Method section.

Line119, (Line121-122): Statements of consent for publication of the individual who appeared in the text was provided as follows: “The individuals in this manuscript have given written informed consent to publish obtained data including images of the participants.”

Changes of Reference in 1st revision were listed below:

Ref #1-11 remained unchanged in 1st revision.

Ref#12-17, and Ref#18-19 were moved to Ref#18-23 and Ref#24-25 in 1st revision.

Ref#20-21 were removed in 1st revision.

Ref#22-24 were moved to Ref#13-14, and Ref#33 in 1st revision.

Ref#12 was added in 1st revision.

Ref#15-17 were added in 1st revision.

Ref#26-30 were added in 1st revision.

Reply to comments of Academic Editor in second revision

Ref#31-32 in 1st revision were removed, because these two literatures were unnecessary in second revision.

Supporting File, that contains detail contents of Table 1, was uploaded as S Table. In this file, unit, number of specimens, mean, median, standard deviation, standard error, minimum, maximum, coefficient of variation for each muscle were presented.

Following marks, Line**-**, (Line**- **), suggests Line in original manuscript, and (Line in first revision).

Reply to comments of Reviewer #1, #2, and #3 in first revision and second revision

Reviewer #1: in the first revision

General comments:

Specific comments:

Response of comment #1): Line105, (Line112-113): Information about recruitment of participant was stated in the Method section as follows: “Subjects were recruited by announcements on posters which were exhibited in a student bulletin board.”

Response of comment #2): Table 1 was removed.

Response of comment #3): Line110, (Line237-238): Normality of the data was moved to statistics section.

Response of comment #4): P5, L122: Number of participants was removed.

Response of comment #5): P5, L128: the reference to the Figure 1 is not related to the text

(Line 133), “The temperature and humidity in the laboratory were maintained at 25°C and 30-40%, respectively”.

Response of comment #6): Line128, (Line132): Reference of (Figure.1) was moved up to adequate location.

Response of comment #7): Line 167-171, (Line170-179): The prove positioning and location on the muscle belly was stated specifically as follows: “The location of a probe to be applied on the muscle belly of the medial and lateral gastrocnemius, soleus and tibialis anterior muscles was adjusted in the designated position (the medial and lateral portions of the gastrocnemius muscle at 30 % of the proximal left lower leg, the soleus muscle at 50 % of the left lower leg, and the tibialis anterior muscle at 30 % of the proximal left lower leg [2, 5]) for individual subject. On the most prominent part of the muscle belly, the rectangular shape skin mark with adhesive tape was attached prior to the measurement, and the probe was placed in the rectangular mark (Fig 3a, 3b). Since orientation of muscle fascicles of the gastrocnemius, soleus is oblique by their pennated structure and is redundant due to muscle relaxation, prove setting was aligned to anatomical muscle shortening direction.”

P7: Did the participants remain in the same position for the elastography measurements? It is not clear that the surface scanned was easily accessible for the evaluator according the image provided.

Response of comment #8): Line 142, (Line155-156): “Participants remained in the same position sitting in a chair from the beginning until the end of the elastography measurement, except 3 minutes leg elevation.” This statement was added at the end of “Elevation of the lower limb”.

Response of comment #9): Line168-171, (Line170-179): A prove positioning and location of the muscle belly was stated specifically as follows: “The location of a probe to be applied on the muscle belly of the medial and lateral gastrocnemius, soleus and tibialis anterior muscles was adjusted in the designated position (the medial and lateral portions of the gastrocnemius muscle at 30 % of the proximal left lower leg, the soleus muscle at 50 % of the left lower leg, and the tibialis anterior muscle at 30 % of the proximal left lower leg [2, 5]) for individual subject. On the most prominent part of the muscle belly, the rectangular shape skin mark with adhesive tape was attached prior to the measurement, and the probe was placed in the rectangular mark (Fig 3a, 3b). Since orientation of muscle fascicles of the gastrocnemius, soleus is oblique by their pennated structure and is redundant due to muscle relaxation, prove setting was aligned to anatomical muscle shortening direction.”

Response of comment #10): Line203, (Line209-212): Statement of traceability of EMG data during elastography measurement for the evaluator was added as follows: “During shear wave velocity measurement by ultra-sonography, EMG data was continuously displayed for examiner and subject on PC screen, suggesting the meaning of appearance of muscle activation waves for ensuring proper relaxation.”

P9, L235: Replace “the risk rate was set at” by “level of significance was set at”

Response of comment #11): Line235, (Line246): “the risk rate was set at” was replaced by “level of significance was set at” as suggested.

P 11 L283- 297: In the discussion, the values obtained should not be repeated. The results should rather be summarized and discussed according the current literature.

Response of comment #12): L283- 297, (Line287-376): All repetitions on reported results were removed from the Discussion

Response of comment #13): Line97, (Line102-106): The hypothesis was added by providing specific pathology in the lower leg muscles.

Response of comment #14): Line333-335, (Line332-359): The hypothesized mechanism was provided in two points of view, i.e. one is change of leg muscle fibers and the other is change of extra-cellular space in the compartment.

Reviewer #2: in the first revision

The reviewer has thoroughly reviewed the manuscript for scientific content, manuscript construction, and general impact. This study seems a sound one but needs to be redefined, especially in terms of novelty, hypothesis, and methodology. The main issues are listed below.

Introduction

The authors consider that the clinical importance of the present study is related to compartment syndrome, it would be better to measure intramuscular pressure instead of shear wave velocity.

Response of comment #1): Line97, (Line102-106): The hypothesis was added by providing specific pathology in the lower leg muscles as follows: “We hypothesized that the shear wave velocity of the medial and lateral gastrocnemius, soleus and tibialis anterior muscle would increase with time in the resting leg position after 2 hours of sitting and decrease with subsequent leg elevation. Then we considered the mechanism of the velocity change with two points of view, i.e. one is change of leg muscle fibers and the other is change of extra-cellular space in the compartment.”

Response of comment #2): Line91, (Line91-96): In the middle of Introduction, estimated muscle condition during a prolonged sitting was listed by mentioning literature as follows: “There are several clinical conditions that cause lower leg symptom during a prolonged sitting, i.e. occupational leg edema after prolonged standing and sitting [12], thrombophlebitis of lower leg during long flight [13, 14] and fluid accumulation in the leg during long-distance bus travel [15]. To solve mechanism of prolonged sitting in lower leg, plethysmographic measurement for haemodynamics of calf veins or bioelectrical impedance analysis for tissue fluid has been performed [16, 17], but no measurement of resting leg muscles by shear wave elastography has been reported.”

Response of comment #3): Line329-, (Line332-359): In Discussion, to verify hypothesis about venous pressure and impact on muscle, speculated pathology occurred in the lower leg muscles was stated with providing literature as follows: “Increase of muscle stiffness in active muscle conditions is reported to have several causes, i.e. reflex-mediated stiffness by prolonged muscle contraction after repetitive task which lead to the release of metabolites and activate nociceptors [26, 27], temporary increase of muscle stiffness after repetitive muscle contraction due to static or dynamic exercises, which is reflected by increased intramuscular pressure associated with increases in intramuscular blood flow [28, 29], or static stretch of the muscles by which muscle stiffness increase proportionally to the muscle length [6]. Increase of muscle stiffness in resting muscle conditions is reported to have causes, i.e. increasing collagen mass in muscles by aging and spastic conditions with neuromuscular diseases [7, 30].” “On the other hand, although no measurement by shear wave elastography has been performed, the another suspected causes of increase in muscle stiffness in resting muscles are reported to be fluid retention in the leg due to occupational leg edema after prolonged standing and sitting [12], long-haul flight simulation of sitting 4 to 12 hours [16], and the effect of 4 ours sitting and calf activity [17]. Belczak et al (2018) reported significant increase in leg volume by volumetric measurement using water displacement technique [12]. Mittermayr et al (2007) reported increased lower leg volume by plethysmographic measurement using an optoelectronic scanner system, suggesting the phenomena was due to extravascular fluid shifts in the lower leg. They also measured by ultrasonography the cross-sectional diameter of calf veins, there was no significant change in the diameter by 4 to 12 hours sitting [16]. Singh et al (2017) reported increased leg volume estimated by calf circumference and by bioelectrical impedance analysis. In their study, they suggested two points, first increase in immediate intravascular leg fluid due to hydrostatic pressure and a loss of leg muscle tone, second retention of extravascular fluid in the legs by hydrostatic pressure after 4 hours sitting [17].”

Response of comment #3) cont’d: Line344, (Line357-359): The following summarized statements was inserted. “Based on literature and result of our study, increase in shear wave velocity with prolonged sitting does not come from change of leg muscle fibers or connective tissue but may come from increase in intra-compartment pressure of the lower leg due to fluid retention in extra-cellular space.”

Response of comment #4): Line347, (Line371-374): Clinical importance was stated about detection and treatment of leg edema with measurement of shear wave velocity by non-invasive ultrasonography as follows: “The results of this study have clinical importance in the possibility of prevention of muscle stiffness and edema of the lower legs during long time flight or evacuation in the car as refugees after huge earth quake. Measurement of shear wave velocity by non-invasive ultrasonography help people know the extent of leg edema in timely fashion.”

Methods

The authors report 3ρVs2 because they regarded the muscles as isotropic (Line 190-). However, this is wrong (although the authors are aware).

The ICC in this study is smaller than that of previous studies (ICC > 0.9).

The authors stated that no muscle contraction was observed to occur through EMG recording. Please explain in more detail what value the authors determined to be no muscle contractions.

Response of comment #5): Line167-171, (Line170-179): Detail placement of probe and location on the muscle belly was stated in the text as follows: “The location of a probe to be applied on the muscle belly of the medial and lateral gastrocnemius, soleus and tibialis anterior muscles was adjusted in the designated position (the medial and lateral portions of the gastrocnemius muscle at 30 % of the proximal left lower leg, the soleus muscle at 50 % of the left lower leg, and the tibialis anterior muscle at 30 % of the proximal left lower leg [2, 5]) for individual subject. On the most prominent part of the muscle belly, the rectangular shape skin mark with adhesive tape was attached prior to the measurement, and the probe was placed in the rectangular mark (Fig 3a, 3b). Since orientation of muscle fascicles of the gastrocnemius, soleus is oblique by their pennated structure and is redundant due to muscle relaxation, prove setting was aligned to anatomical muscle shortening direction.”

Response of comment #6): Line203-204, (Line212-213): Statement of EMG activity recording was corrected as follows: “Appearance of EMG activity was determined by observation of action potentials greater than 2SD amplitude of baseline.”

Discussion

Response of comment #7): Line329-, (Line332-359): We added in Discussion hypothesized mechanism with two points of view, i.e. one is change of leg muscle fibers and the other is change of extra-cellular space in the compartment.

Conclusion

Intramuscular pressure was not directly measured in this paper. Thus, the current conclusion is not based on the data of the present study.

Response of comment #8): Line384-391, (Line403-407): Conclusion was revised based on the result of increase in shear wave velocity and speculated pathology in the lower leg muscle as follows: however, in second revision, based on comment from Reviewer #3, Conclusion was finally revised as follows:

“Shear wave velocity of the lower leg muscles increased with time in 2 hours sitting, and decreased with subsequent leg elevation. Based on the report that the change in the shear wave velocity was proportional to the internal pressure of the leg muscle compartment in turkey models, it is estimated that increase in shear wave velocity with prolonged sitting may come from increase in intra-compartment pressure of the lower leg due to fluid retention in extra-cellular space of the compartment.”

Response of comment #9): Line43-44, (Line44-45): Conclusion in Abstract was revised based on Conclusion in the Text as follows: “and it is assumed that the increase of shear wave velocity in the 2-hour seated leg is due to fluid retention in extra-cellular space of the compartment.”

Reviewer #3: in first revision

1) By considering the limited sample size, I would add “a proof-of-concept study” at the end of the title.

Response of comment #1): Line1, (Line2): “a proof-of-concept study” was added in the title as suggested.

2) Abstract, L38-39: There is repetitive information on statistics.

Response of comment #2): Line30-39, (Line39-40): The repetitive part was removed and data in parenthesis was corrected.

Response of comment #3 and 4): Line75-76, (Line72-79): The sentence “On the contrary, if the density of muscles is not uniform, it is expressed in terms of the anisotropic modulus (anisotropic E= ρVs2) [2,7].” was removed to avoid confusion. And the paragraph was revised as follows, “Regarding the difference between elastic modulus and shear wave velocity, Eby (2013) found that the equation E=3ρVs2 (E: elastic modulus kPa, Vs: shear wave velocity m/s, ρ: muscle density 1000 kg/m3) holds if the density of the muscle is assumed to be uniform, in case of measurement along longitudinal muscle fiber direction [4]. On the contrary, if the density of muscles is not uniform, in case of measurement along transverse or oblique muscle fiber direction, it is expressed in terms of the anisotropic modulus (E=ρVs2) [2,7]. In researches of the shear wave velocity in the leg muscles at a resting state (ankle position around the slack angle without muscle contraction), the muscle tissue was measured regarding as the isotropic modulus (E= 3ρVs2) [2,5,7-9].”

5) L97: Your hypothesis is empty and not based on strong rationales. This should be improved. Why prolonged sitting would change muscle stiffness?

Response of comment #5): Line97, (Line102-106): The hypothesis was added by providing specific pathology in the lower leg muscles as follows: “We hypothesized that the shear wave velocity of the medial and lateral gastrocnemius, soleus and tibialis anterior muscle would increase with time in the resting leg position after 2 hours of sitting and decrease with subsequent leg elevation. Then we considered the mechanism of the velocity change with two points of view, i.e. one is change of leg muscle fibers and the other is change of extra-cellular space in the compartment.”

6) Table 1 is useless and should be removed.

Response of comment #6): Table 1 was removed and detail statement of this result was provided in the Subjects (Line 114-117).

7) L159: “precision”.

Response of comment #7): Line159: typo error was corrected

8) L186 and 188: What is a “shear wave verbosity” and what is “a constant condition”?

Response of comment #8): Line186: “verbosity” was corrected for “velocity”, and “a constant” was removed as suggested.

9) L203: What is the output of the Web-1000 system?

Response of comment #9): Line203, (Line207-209): The sentence was corrected as follows: “The electromyography data was recorded at 2000 Hz and collected to a polygraph system of Web-1000 (Nihon Koden Ltd., Tokyo, Japan), then analyzed using LabChart version 7 (AD Instruments, Tokyo, Japan).”

10) Discussion: Remove all repetitions on reported results, this is useless.

Response of comment #10): L283- 297, (Line288-374): All repetitions on reported results were removed from the Discussion

11) L309 and elsewhere: Correct “verbosity”.

Response of comment #11): Line310: All typo error was corrected.

Response of comment #12): Line309-320, (Line315-320): These two paragraphs were revised to state the effect of hydrostatic pressure in the low leg with sitting position to avoid confusions as follows: “In sitting position, venous pressure of popliteal vein at the popliteal fossa and pressure of saphenous vein at the medial malleolus were reported to be 23.9 mmHg (3.17 kPa) and 54.2 mmHg (7.2 kPa), respectively [25]. Considering the location of shear wave velocity measurements in the tibialis anterior and gastrocnemius muscles in the sitting position, it can be inferred that hydrostatic pressure is equally applied to the tibialis anterior and gastrocnemius muscles through the leg veins for two hours.”

13) L317: You are overstretching the interpretation of your results since you did not measure the intramuscular pressure; consequently you cannot say that this is “in accordance with”.

Response of comment #13): Line317: This paragraph was removed.

14) L327: The muscle length has certainly nothing to do with shear wave speed; you are likely referring to the muscle stretching.

Response of comment #14): Line327, (Line327): We consider the statement of muscle length in this context was correct.

15) L328: “nature of muscle”, can you be more descriptive and support your hypothesis based on known histopathology analysis?

Response of comment #15): Line328, (Line328): The phrase “nature of muscle” was replaced by “pennation angle of muscle fibers or muscle fiber length”

Response of comment #16): Line329-, (Line332-359): To verify hypothesis about venous pressure and impact on muscle, speculated pathology occurred in the lower leg muscles was stated with providing literature as follows: “Increase of muscle stiffness in active muscle conditions is reported to have several causes, i.e. reflex-mediated stiffness by prolonged muscle contraction after repetitive task which lead to the release of metabolites and activate nociceptors [26, 27], temporary increase of muscle stiffness after repetitive muscle contraction due to static or dynamic exercises, which is reflected by increased intramuscular pressure associated with increases in intramuscular blood flow [28, 29], or static stretch of the muscles by which muscle stiffness increase proportionally to the muscle length [6]. Increase of muscle stiffness in resting muscle conditions is reported to have causes, i.e. increasing collagen mass in muscles by aging and spastic conditions with neuromuscular diseases [7, 30].”

Response of comment #16) cont’d: “On the other hand, although no measurement by shear wave elastography has been performed, the another suspected causes of increase in muscle stiffness in resting muscles are reported to be fluid retention in the leg due to occupational leg edema after prolonged standing and sitting [12], long-haul flight simulation of sitting 4 to 12 hours [16], and the effect of 4 ours sitting and calf activity [17]. Belczak et al (2018) reported significant increase in leg volume by volumetric measurement using water displacement technique [12]. Mittermayr et al (2007) reported increased lower leg volume by plethysmographic measurement using an optoelectronic scanner system, suggesting the phenomena was due to extravascular fluid shifts in the lower leg. They also measured by ultrasonography the cross-sectional diameter of calf veins, there was no significant change in the diameter by 4 to 12 hours sitting [16]. Singh et al (2017) reported increased leg volume estimated by calf circumference and by bioelectrical impedance analysis. In their study, they suggested two points, first increase in immediate intravascular leg fluid due to hydrostatic pressure and a loss of leg muscle tone, second retention of extravascular fluid in the legs by hydrostatic pressure after 4 hours sitting [17]”

Response of comment #16) cont’d: Line344, (Line357-359): The following summarized statements was inserted. “Based on literature and result of our study, increase in shear wave velocity with prolonged sitting does not come from change of leg muscle fibers or connective tissue but may come from increase in intra-compartment pressure of the lower leg due to fluid retention in extra-cellular space.”

Response of comment #17): Line342, (Line346-355): Providing literature, statement of plethysmographic measurement and bioelectrical impedance analysis for leg edema was added in Discussion.

18) Conclusion: Here again you are overstretching the interpretation of your results; no internal pressure has been reported.

Response of comment #18): Line384-391, (Line406-411): Conclusion was revised based on the result of increase in shear wave velocity and speculated pathology in the lower leg muscle as follows: “Shear wave velocity of the lower leg muscles increased with time in 2 hours sitting, and decreased with subsequent leg elevation. It has been reported that the change in the shear wave velocity was proportional to the internal pressure of the leg muscle compartment. Increase in shear wave velocity with prolonged sitting may come from increase in intra-compartment pressure of the lower leg due to fluid retention in extra-cellular space of the compartment.”

19) L391: “evacuation in the car”, what do you mean?

Response of comment #19): Line391, (Line371-374): We added statement of evacuation in the car as refugees after huge earth-quake as follows: “The results of this study have clinical importance in the possibility of prevention of muscle stiffness and edema of the lower legs during long time flight or evacuation in the car as refugees after huge earth quake. Measurement of shear wave velocity by non-invasive ultrasonography help people know the extent of leg edema in timely fashion [14, 31]”

Reviewer #3 in second revision

No decision can be made unless a new "responses to reviewers" document is made with clear color-coded changes.

Response of comment #1): Response to reviewer’s comments was clearly indicated in rebuttal letter as suggested.

Response of comment #2): A different color code was used for each reviewer in the text; brown for reviewer #1, green for reviewer #2, blue for reviewer #3.

Response of comment #3): To avoid over-stretching statement in The conclusion, the sentence "It has been reported that the change in the shear wave velocity was proportional to the internal pressure of the leg muscle compartment.", was revised as follows (Line 407-411): “Based on the report that the change in the shear wave velocity was proportional to the internal pressure of the leg muscle compartment in turkey models, it is estimated that increase in shear wave velocity with prolonged sitting may come from increase in intra-compartment pressure of the lower leg due to fluid retention in extra-cellular space of the compartment.”

Response of comment #4): Typo errors were corrected.

In second revision, we added a sentence at the last paragraph of Discussion. L374, It is as follows: “In such cases, it is important to know that simple elevation of lower leg for 3 minutes may reduce leg edema.”

Attachment

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Click here for additional data file.^{(43.4KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0251532.r005

Decision Letter 2

Guy Cloutier

28 Apr 2021

Effect of prolonged sitting immobility on shear wave velocity of the lower leg muscles in healthy adults: a proof-of-concept study

PONE-D-20-36699R2

Dear Dr. Aoki,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Guy Cloutier, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0251532.r006

Acceptance letter

Guy Cloutier

30 Apr 2021

PONE-D-20-36699R2

Effect of prolonged sitting immobility on shear wave velocity of the lower leg muscles in healthy adults: a proof-of-concept study

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Shear wave velocity of the lower leg muscles (n = 24).

(DOCX)

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Data Availability Statement

All relevant data are within the paper and its Supporting Information file.

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PERMALINK

Effect of prolonged sitting immobility on shear wave velocity of the lower leg muscles in healthy adults: A proof-of-concept study

Kumiko Okino

Mitsuhiro Aoki

Masahiro Yamane

Chikashi Kohmura

Roles

Abstract

Objective

Materials and methods

Results

Conclusions

Introduction

Materials and methods

Subjects

Experimental protocol

Fig 1. Experimental design.

Sitting posture

Fig 2.

Elevation of the lower limb

Measurement of shear wave velocity

Fig 3.

Fig 4. Image of shear wave elastography of lower leg muscles.

Surface electromyography

Reliability analysis

Sample size

Statistical analysis

Result

Change in the shear wave velocity of the leg muscles with time

Table 1. Shear wave velocity of LG, MG, SOL, and TA over time.

Fig 5. Shear wave velocity of the lateral gastrocnemius.

Fig 6. Shear wave velocity of the medial gastrocnemius.

Fig 7. Shear wave velocity of the soleus.

Fig 8. Shear wave velocity of the tibialis anterior muscle.

Comparison of shear wave velocity between muscles immediately after the start of sitting

Discussion

Research limitations

Measurement of muscle at rest

Measurement for adult male

Measurement up to 2 hours

Venous hemodynamics

Conclusions

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Guy Cloutier

Roles

Author response to Decision Letter 0

Decision Letter 1

Guy Cloutier

Roles

Author response to Decision Letter 1

Decision Letter 2

Guy Cloutier

Roles

Acceptance letter

Guy Cloutier

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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