Accepted Manuscript

1

Type of manuscript: Short communication

2

Title: Reliability of ultrasonographic measurement of muscle and tendon morphology

3using the Overlapping Images Method

4Names of authors: Akane Yoshimura*1,2, Masaaki Honda 3, Shinshiro Mineta 1,4,

5

Kenichiro Murata 1,5, Norikazu Hirose 3

6

Affiliations and Mailing addresses:

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1 Graduate School of Sport Sciences, Waseda University, 3-4-1 Higashifushimi

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Nishitokyo-shi Tokyo 202-0021 Japan (Affiliation where the experiment was

9conducted)

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2 Faculty of Education and Integrated Arts and Sciences, Waseda University, 1-6-1

11Nishiwaseda Shinjuku-ku Tokyo 169-8050 Japan (Current affiliation)

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3 Faculty of Sport Sciences, Waseda University, 2-579-15 Mikajima Tokorozawa-shi

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Saitama 359-1192 Japan14

4 Department of Health and Sports Science, Fukuyama Heisei University, 117-1 Masato

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Kamiiwanari Miyuki-cho Fukuyama-shi Hiroshima 720-0001 Japan (Current affiliation)

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5 Yokohama Tama-Plaza Sports Clinic, 2-14-9 Utsukushigaoka Aoba-ku Yokohama-shi

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Kanagawa 225-0002 Japan (Current affiliation)

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Number of figures: 3

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Number of tables: 3

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Brief running title (70 characters or less): Overlapping Images Method for triceps

21

surae

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Corresponding author’s e-mail address*:

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Name: Akane Yoshimura

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E-mail address: ayoshimura@aoni.waseda.jp

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Affiliation and mailing address: Graduate School of Sport Sciences, Waseda

26

University; Faculty of Education and Integrated Arts and Sciences Waseda

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University

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Abstract

35

Problem addressed: Previous attempts have been made to superimpose ultrasound36

images (Overlapping Images Method). The merit of this method is that it enables

37

capturing the entire morphology of the muscle and tendon in the longitudinal direction

38

beyond the width of the ultrasound probe. However, the division points between tissues

39

were identified visually, and the reliability of the measurement was not clarified.

40

Experimental approach: This study identified the division points for the overlapping

41

images method using the following two procedures: (1) visual-only identification and

42(2) a combination of visual and custom-built software-based identification. Finally, the43

reliability of the measurements for each procedure was examined by estimating the44

intra- and inter-rater correlation coefficients (ICC 1.1, ICC 2.1). The length of the45

Achilles tendon, muscle-tendon unit, and gastrocnemius muscle was measured using the46

overlapping images method for 19 volunteers. Main results and findings: The ICC 1.1

47

and ICC 2.1 scores for visual identification alone ranged between 0.61–0.91 and 0.70–

48

0.93, respectively. The scores for the combination of visual and software-based

49

identification ranged 0.90–0.99 and 0.58–0.96, respectively. Conclusions: The high50

intra- and inter-rater reliability in the overlapping images method was demonstrated in51

almost all measurements of the three tissue lengths and the applicability of overlapping

52

images method was demonstrated. In particular, the intra-rater reliability was better

53

when a combination of visual and custom-built software-based identification was used.

54

Keywords: Achilles tendon, gastrocnemius muscle, ultrasound image, reliability

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Information in Japanese68

タイトル：「オーバーラッピング画像法」による超音波を用いた筋及び腱の形

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態評価の信頼性70

著者名：吉村茜* 1,2, 誉田雅彰 3, 峯田 晋史郎 1,4, 村田 健一朗 1,5, 広瀬統一 3

71

所属：

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1 早稲田大学スポーツ科学研究科（研究実施時の所属）

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2 早稲田大学教育・総合科学学術院（現在の所属）

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3 早稲田大学スポーツ科学学術院

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4 福山平成大学健康スポーツ科学科（現在の所属）

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5 横浜たまプラーザ 運動器スポーツクリニック（現在の所属）

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アブストラクト:78

Problem addressed: 先行研究において、「オーバーラッピング画像法」と呼ば79れる個別の超音波画像を重ね合わせる手法によって、筋や腱の形態を測定する

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試みがされている（Urlando, A., & Hawkins, D. (2007). Med Sci Sports Exerc.,

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39(7): 1147-1152）。この手法のメリットは、プローブ幅に依存せず、長軸方向で82

組織の全長を計測できる点である。しかしながら、先行研究では重ね合わせた83

超音波画像に描出される組織の分岐点について目視で識別されており、この手

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法の信頼性について明確に示されていない。Experimental approach: そこで本85

研究では、（1）目視により識別、（2）目視とカスタマイズしたソフトウェアの86両方による識別、以上 2 つの手順により超音波画像上の組織の分岐点を定義

87

し、それぞれの手順によるオーバーラッピング画像法の検者内及び検者間信頼88性（ICC1.1, ICC2.1）を検討することとした。対象は健常成人 19 名とし、アキ89

レス腱、筋腱部複合部、腓腹筋の全長をオーバーラッピング画像法を使用して90計測した。Main results and findings: 目視による識別に基づく測定において

91

ICC1.1 と ICC2.1 のスコアは、それぞれ 0.61-0.91 と 0.70-0.93 の範囲内であっ92た。一方、目視とソフトウェアの両方による識別に基づく測定において ICC1.1

93と ICC2.1 のスコアは、それぞれ 0.90-0.99 と 0.58-0.96 の範囲内であった。

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Conclusions: オーバーラッピング画像法における検者内及び検者間信頼性はお95

およそ高いものと評価され、応用可能性が示された。特に、目視とソフトウェ

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アの両方による識別に基づく測定では、目視のみの識別の場合と比べて検者内97

信頼性が高かった。

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Introduction
Most previous studies have used ultrasound to evaluate muscle

103

morphology, as it is non-invasive and measurements are performed in real time. It is104

well-known that ultrasound has been widely used to capture morphological105

characteristics in sports sciences. For example, we previously measured muscle

106morphology and extensibility using b-mode ultrasound to investigate the effect of

107

massage interventions before and after the intervention 1,2). In addition, based on the108morphological characteristics reflected on ultrasound images, attempts have been made109

to detect age-related changes in muscle mass in growing children and to explain the110

effects of long-term strength training on muscle mass 3,4). However, a limitation of111

morphological measurements by b-mode ultrasound is that the measurable area depends

112

on the width of the ultrasound probe. Therefore, morphological characteristics generally

113

cannot be measured over an area wider than the width of the ultrasound probe. Although114

the panoramic view function has been used in recent years to overcome this limitation

115

and seems to be reliable and valid 5), devices equipped with this function are limited.

116

Some scientists have attempted to superimpose ultrasound images obtained

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separately by moving the probe longitudinally over the skin of the Achilles tendon and118

gastrocnemius muscle. This method is called the “Overlapping Images Method (OIM)”

119

6,7,8)

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the division points reflected in ultrasound images, such as the proximal and/or distal

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tendon and muscle ends, has not been fully clarified.

122. However, a serious problem is the reliability of this method for visually identifying

Against this backdrop, the present study identified the division point of OIM

123

using the following two procedures: (1) conventional visual-only identification and (2) a124

combination of visual and custom-built software-program-based identification, which125

was conducted out of concern regarding the low reliability of visual-only identification.

126

We then examined the intra- and inter-rater reliabilities of the OIM measurements for127

each procedure.

128

Based on the only report showing that the high intra-rater reliability was

129

achieved for the OIM measurement of Achilles tendon length 7), we hypothesized that130

the OIM measurement would be a reliable method for both procedures. We also131

predicted that the reliability would improve by using a combination of visual and132

software identification.

133

Materials and Methods

134

Study population

135

This study was approved by the Research Ethical Review Committee with

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Human Subjects of Waseda University, and all procedures were performed in137

accordance with the Declaration of Helsinki (approval number: 2016-101). Data138

obtained from nineteen volunteers (12 men and 7 women) was analyzed. All

139

participants were fully informed of the procedure and purpose of the study, and140

provided written informed consent.

141

Procedure

142

Participants were instructed to lie on their backs on a Biodex chair (Biodex

143

System 3; SAKAI Medical Co., Ltd., Tokyo, Japan), with the seat placed approximately

144

parallel to the floor. The right ankle joint was fixed to a plate attached to Biodex, and145

the knees and hips were maintained in neutral positions. When the fixed ankle was146

moved passively with the plate to prevent excessive varus and valgus joint motions, the147

maximum dorsiflexion and plantar flexion ranges of motion of the ankle displayed on148

the Biodex screen were recorded. Simultaneously, an electromagnetic 2-dimensional

149

goniometer (DTS EM-852; SAKAI Medical Co., Ltd., Tokyo, Japan) was attached to150

the skin along the right femur and fibula lines to monitor the knee joint range of motion

151

during the ankle’s passive movement so that the compensation movement could be

152

regulated to within ± 5°.

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Ultrasound measurement

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Ultrasound images of the Achilles tendon, muscle-tendon unit, and155

gastrocnemius muscle were obtained based on the processes of the OIM 6,7,8) with the156

ankle joint in three different positions: neutral, maximum dorsiflexion, and maximum

157

plantar flexion. These processes include the following steps:

158[Step-1] Pieces of cut thread were arranged and stuck with a tape on the skin on

159

the back of the lower leg in a straight line from the center of the calcaneal tuber to the160most proximal end of the gastrocnemius muscle, serving as landmarks at intervals

161

smaller than the width of the ultrasound probe (i.e., approximately 3 cm) (SNiBLE;

162

Konica Minolta, Inc., Tokyo, Japan). (Figure 1).

163[Step-2] The ultrasound probe was placed on the skin over the calcaneal tuber,

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and the first ultrasound image was acquired by visualizing the Achilles tendon insertion

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site and landmarks at both ends of the image.

166[Step-3] The probe was applied over the skin from the calcaneal tuber to the167most proximal end of the gastrocnemius muscle and the ultrasound images were168

acquired by visualizing landmarks at both ends of each image.

169[Step-4] Each image acquired in Step-3 was overlapped based on marker lines,

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and an image showing the overall Achilles tendon, muscle-tendon unit, and171

gastrocnemius muscle was created by superimposing each image (Figure 2) in the172

neutral, maximum dorsiflexion, and maximum plantar flexion positions, respectively.

173[Step-5] Four division points (i.e., the most distal end of the Achilles tendon,174

the muscle-tendon junction of the soleus muscle, the muscle-tendon junction of the

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gastrocnemius muscle, and the most proximal end of the gastrocnemius muscle) in the176

image made in Step-4 were identified in two ways; the first was by the raters’ visual

177

identification, and the second was using custom-built MATLAB-based program

178(MathWorks) in addition to visual identification. In the software identification approach,179

the division points were decided by scanning within regions of interest (ROI) set into

180

MATLAB after importing the ultrasound image into the software program. First, the181

ROI-reference (red square in Figure 3) was placed at the most proximal position at each182

division point, and an ROI-scan range (orange square in Figure 3) was placed at the183

whole division point covering the ROI-reference (step-5-1 in Figure 3). Next, the area

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within the ROI-scan (green square in Figure 3) was scanned while moving from the185

distal to the proximal side (step-5-1 in Figure 3). Finally, texture features in ROI-

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reference and scan were calculated by gray-scale histogram analysis and squared euclid

187

distance (SED), which is an indicator for traditional similarity evaluation 9), between

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ROI-reference and each ROI-scan (step-5-2 in Figure 3). The position where SED

189values were maximum (i.e., the lowest degree of similarity) was defined as the division

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point between tissues.

191[Step-6] The lengths of the Achilles tendon, muscle-tendon unit, and192

gastrocnemius muscle were measured using ImageJ (National Institution of Health)193

based on the definition in Step-5.

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All steps were performed by rater A, whereas only step 5 was performed by all

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three examiners (raters A, B, and C) to examine the inter-rater reliability.

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Statistical analysis

197

In order to examine the intra- and inter-rater reliabilities of OIM measurement198

by visual-only identification and a combination of visual and software identification, the199

ICC 1.1 and ICC 2.1 scores were estimated. ICC 1.1 was calculated using the data

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measured by rater A, whereas ICC 2.1 was conducted using the data measured by raters

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A, B, and C. In addition, based on the definition that the value obtained by visual-only202

identification is the “true value,” the value differences between visual-only and203

combination identification were examined using the paired t-test or Wilcoxon signed-

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rank test following the Shapiro–Wilk normality test to investigate the validity of the205

combination identification. The level of significance was set as p < .05. IBM SPSS

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Statistics for Windows, Version 25.0. (IBM Corp., Armonk, NY, IBM Corp.) was used

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for all the statistical analyses.

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Results
The age, height, and weight of the 19 participants were 23.36±2.54 years,
164.36 ± 8.05 cm, 60.76 ± 10.57 kg (mean ± standard), respectively.
Tables 1 and 2 show the evaluated ICC scores, and Table 3 shows each tissue212

length and the differences in absolute values between visual-only and combination

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identification. The ICC 1.1 and ICC 2.1 scores by the visual-only identification were214

0.61–0.91 and 0.70–0.93, respectively. These scores by the combination identification

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ranged 0.90–0.99 and 0.58–0.96, respectively. There were significant differences216

between visual-only and combination identification in the values of the Achilles tendon

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at the neutral and maximum plantar flexion positions.

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Discussion

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We investigated the intra- and inter-rater reliabilities of visual-only and220

combination identification in OIM by comparing the lengths of the Achilles tendon,

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muscle-tendon unit, and gastrocnemius muscle measured based on ultrasound image

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divisions. Based on previously reported criteria: poor, < 0.40; fair, 0.40–0.59; good,223

0.60–0.74; and excellent ≥ 0.75 10,11), the intra- and inter-rater reliability was “good” or224“excellent” for both visual-only and combination identification in the present study. In

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particular, the OIM determined by combination identification showed a high intra-rater

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reliability of the length measurements of the Achilles tendon, muscle-tendon unit, and227

gastrocnemius muscle, which supported our hypothesis. To investigate the validity of228

the combination identification, we also examined the differences between visual-only229

and combination identification based on the definition that the value determined by the230

visual-only identification is the “true value.” There were no significant differences231

between visual-only and combination identification, except in the length of the Achilles

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tendon at the neutral and maximum-plantar flexion positions. The findings of the233

present study suggest that OIM is applicable in the field of sports science as a method234

for investigating the influence of massage intervention, training, or growth in children

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on the overall length of muscles and tendons.

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Notably, the ICC score on the gastrocnemius muscle measurement at neutral

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position was lower than that of other measurements. We identified four division points

238(the distal end of the Achilles tendon, soleus muscle-tendon junction, gastrocnemius

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muscle-tendon junction, and gastrocnemius proximal end). Among them, the black-and-240

white ultrasound images were less clear at the proximal end of the gastrocnemius

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relative to the gastrocnemius length measurement than at the other division points.

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Because we determined the division point using the MATLAB program grayscale

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histogram in the combination identification, black-and-white ultrasound image

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ambiguity may make grayscale histogram identification difficult, reducing the245measurement reliability. This is a limitation of the combination identification.

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Significant differences in the Achilles tendon length measurements were found between

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visual-only and combination identification at neutral and maximum dorsiflexion

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positions using the paired t-test. This result suggests that it may be somewhat more249

difficult to identify the division points of the tissues involved in measuring the Achilles

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tendon length (i.e., the distal end of the Achilles tendon and soleus muscle-tendon

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junction). The clarity of the distal end of the Achilles tendon is likely to vary depending252

on the ultrasound incidence angle, and some of the images acquired in the present study

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were unclear. Therefore, it is important to clearly reflect the distal end of the Achilles

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tendon in ultrasound image acquisition to guarantee the high reliability and validity of255

the OIM measurement.

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Conclusion

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This study examined the reliability of OIM by incorporating both visual-only258

and combination identification procedures. This demonstrates that the high intra- and259

inter-rater reliability can be obtained in almost all length measurements of the AT,

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muscle-tendon unit, and gastrocnemius muscle at three different positions (neutral,

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maximum dorsiflexion, and maximum plantar flexion positions). In particular, OIM

262using combination identification showed a higher intra-rater reliability. The applicability263

of the overlapping imaging method is demonstrated by the findings of the present study.

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Acknowledgements: No outside funding was received for this study.

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Conflicts of interest: None in particular.

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Author contributions

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A.Y.: Conceptualization, Data curation, Writing-Original Draft preparation,

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Investigation. M.H.: Methodology, Software, Writing-Reviewing and Editing. S.M.:

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Data curation, Investigation, Writing-Reviewing and Editing. K.M.: Data curation,

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Investigation. N.H.: Conceptualization, Writing-Reviewing and Editing.

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References

279

1) Yoshimura A, Inami T, Schleip R, Mineta S, Shudo K, and Hirose N. 2019. Effects

280

of Self-myofascial Release Using a Foam Roller on Range of Motion and

281

Morphological Changes in Muscle: A Crossover Study. Journal of Strength and282

Conditioning Research 35: 2444-2450. doi: 10.1519/JSC.0000000000003196.

283

2) Yoshimura A, Schleip R, and Hirose N. 2020. Effects of Self-Massage Using a284

Foam Roller on Ankle Range of Motion and Gastrocnemius Fascicle Length and285

Muscle Hardness: A Pilot Study. J Sport Rehabil 29: 1171-1178. doi:286

10.1123/jsr.2019-0281.

287

3) Kanehisa H, Ikegawa S, Tsunoda N, and Fukunaga T. 1995. Strength and cross-288

sectional areas of reciprocal muscle groups in the upper arm and thigh during289

adolescence. Int J Sports Med 16: 54-60. doi: 10.1055/s-2007-972964.

2904) Kawakami Y, Abe T, Kuno SY, Fukunaga T. 1995. Training-induced changes in291

muscle architecture and specific tension. Eur J Appl Physiol Occup Physiol 72: 37-292

43. doi: 10.1007/BF00964112.

2935) Valera-Calero JA, Ojedo-Martín C, Fernández-de-Las-Peñas C, Cleland JA, Arias-

294

Buría JL, Hervás-Pérez JP. 2021. Reliability and Validity of Panoramic Ultrasound295

Imaging for Evaluating Muscular Quality and Morphology: A Systematic Review.

296

Ultrasound Med Biol 42: 185-200. doi: 10.1016/j.ultrasmedbio.2020.10.009.

2976) Urlando A, and Hawkins D. 2007. Achilles tendon adaptation during strength298

training in young adults. Med Sci Sports Exerc 39: 1147-1152. doi:299

10.1249/mss.0b013e31805371d1.

3007) Geremia JM, Bobbert MF, Casa Nova M, Ott RD, Lemos FdeA, Lupion RdeO,

301

Frasson VB, and Vaz MA. 2015. The structural and mechanical properties of the302

Achilles tendon 2 years after surgical repair. Clin Biomech Bristol, Avon 30: 485-

303

492. doi: 10.1016/j.clinbiomech.2015.03.005.

3048) Freire B, Dias CP, Goulart NB, de Castro CD, Becker J, Gomes I, and Vaz M A.

305

2017. Achilles tendon morphology, plantar flexors torque and passive ankle

306

stiffness in spastic hemiparetic stroke survivors. Clin Biomech Bristol, Avon 41: 72-307

76. doi: 10.1016/j.clinbiomech.2016.12.004.

3089) Ghaffari A, and Fatemizadeh E. 2015. RISM: Single-Modal Image Registration via

309

Rank-Induced Similarity Measure. IEEE Trans Image Process 24: 5567-5580. doi:

310

10.1109/TIP.2015.2479462.

311

10) Cicchetti DV, and Sparrow SA. 1981. Developing criteria for establishing interrater

312

reliability of specific items: applications to assessment of adaptive behavior. Am J

313

Ment Defic 86: 127-137.

31411) Leung H, Latella C, Lamon S, and Hendy AM. 2018. The Reliability of315

Neurological Measurement in the Vastus Medialis: Implications for Research and316

Practice. Front Psychol 9: 1857. doi: 10.3389/fpsyg.2018.01857.

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Figure legends

331
332

Figure 1. Cut thread arranged on the skin from the calcaneal tuber to the proximal end

333of the gastrocnemius muscle.

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336
337
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339

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Figure 2. Images superimposed for length measurement of the Achilles tendon, muscle-

342

tendon unit, and gastrocnemius muscle.

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Figure 3. Process in [Step-5] using custom-built MATLAB-based program.

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ROI = regions of interest, SED = squared euclid distance

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Tables363

Table 1. Intraclass correlation coefficient scores for intra-rater reliability in visual-only364

and combination of visual and software identification (ICC 1.1).
Visual-only

Combination

identification

identification

NeutralAchilles tendon

0.91

0.99

position

Muscle-tendon unit

0.84

0.98

Gastrocnemius muscle

0.69

0.99

Max-

Achilles tendon

0.76

0.95

dorsiflexion

Muscle-tendon unit

0.89

0.98

Gastrocnemius muscle

0.81

0.90

Max-

Achilles tendon

0.70

0.95

plantar flexion

Muscle-tendon unit

0.61

0.96

Gastrocnemius muscle

0.90

0.94

365

Max-dorsiflexion = maximum dorsiflexion position; max plantar flexion = maximum

366

plantar flexion position.

367

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Table 2. Intraclass correlation coefficient scores for inter-rater reliability in visual-only369

identification and combination of visual and software identification (ICC 2.1).
Visual-only

Combination

identification

identification

NeutralAchilles tendon

0.85

0.91

position

Muscle-tendon unit

0.84

0.96

Gastrocnemius muscle

0.79

0.58

Max-

Achilles tendon

0.77

0.92

dorsiflexion

Muscle-tendon unit

0.89

0.89

Gastrocnemius muscle

0.93

0.82

Max-

Achilles tendon

0.70

0.91

plantar flexion

Muscle-tendon unit

0.72

0.83

Gastrocnemius muscle

0.88

0.89

370

Max dorsiflexion = maximum dorsiflexion position; max plantar flexion = maximum

371

plantar flexion position.

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373

374

Table 3. Length of each tissue and differences in the absolute values between in visual-375

only identification and combination of visual and software identification (cm).
Visual-only

Combination

Value

identification

identificationdifferences

7.11±1.93

7.47±1.91

0.60±0.73<.0113.50±2.2813.35±2.48

0.71±0.94.95

Gastrocnemius muscle 19.34±2.24

19.37±2.63

1.20±1.76

.91

Max-

Achilles tendon

7.67±1.75

7.80±1.55

0.56±0.82

.30

dorsi

Muscle-tendon unit

13.07±2.30

13.31±2.39

0.68±0.78.13

flexion

Gastrocnemius muscle 20.11±2.39

20.17±2.53

0.78±0.87

.67

Max-

Achilles tendon

6.94±1.74

7.21±1.60

0.68±1.01<.01

plantarMuscle-tendon unit

13.48±2.08

13.74±1.98

0.79±1.26.19

flexion

Gastrocnemius muscle 17.37±2.48

17.71±2.95

0.92±1.06.06

NeutralAchilles tendon

position Muscle-tendon unitp376

Values are presented as the mean ± standard deviation. Max dorsiflexion = maximum

377

dorsiflexion position; max plantar flexion = maximum plantar flexion position.

378
379