Tibial nerve stiffness is related to maximum angle of ankle dorsiflexion

22 The maximum angle of ankle dorsiflexion is affected by the triceps surae muscle stiffness and 23 stretch tolerance, which may be strongly reflected by the tibial nerve stiffness. However, no study 24 has evaluated the triceps surae muscle and tibial nerve stiffness simultaneously or clarified their 25 association with the maximum angle. The purpose of this study was to investigate the association 26 between the maximum angle and both the stiffness . Forty-one healthy adults participated . The 27 shear wave velocities of the triceps surae muscles and tibial nerve were measured at 5°, 15°, and 28 25° ankle dorsiflexion. Multiple linear regression analysis was performed using the forced entry 29 method, specifying the shear wave velocities of the four tissues as the independent variables and 30 the maximum angle as the dependent variable. This analysis was performed at each angle where 31 the shear wave velocity was measured. Multiple linear regression analysis was also performed 32 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 22, 2025. ; https://doi.org/10.1101/2025.07.20.665743doi: bioRxiv preprint using the stepwise method, specifying the shear wave velocit ies of the tibial nerve at the three 33 angles as the independent variables and the maximum angle as the dependent variable. Using the 34 forced entry method, the shear wave velocity of the tibial nerve at each angle was significantly 35 negatively associated with the maximum angle, whereas those of the muscles were not. Using the 36 stepwise method, only the shear wave velocity of the tibial nerve at 25° was significantly 37 negatively associated with the maximum angle. These results suggest that the tibial nerve stiffness 38 in a greatly lengthened position determines the maximum angle of ankle dorsiflexion. 39 40 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 22, 2025. ; https://doi.org/10.1101/2025.07.20.665743doi: bioRxiv preprint 1. Introduction 41 The maximum angle of ankle dorsiflexion is an index of joint flexibility that is frequently used in 42 clinical assessments. It is associated with impaired balance and walking ability in elderly 43 individuals (Chiara Mecagni et al., 2000) and is a risk factor for patellar tendinopathy (Backman 44 and Danielson, 2011; Malliaras et al., 2006) . Thus, it is important to investigate the factor s that 45 determine the maximum angle of ankle dorsiflexion. 46 Conventionally, the maximum joint angle is considered to be affected by muscle 47 stiffness and stretch tolerance (Magnusson et al., 1997). Recently, nerve stiffness was suggested 48 to be associated with stretch tolerance (Andrade et al., 2018) . Although some previous studies 49 have reported a correlation between muscle or nerve stiffness and the maximum angle of ankle 50 dorsiflexion, few studies have measured muscle and nerve stiffness simultaneously and 51 investigated their correlations with the maximum angle of ankle dorsiflexion . The medial and 52 lateral gastrocnemius muscle stiffness of healthy young males at 14° of ankle dorsiflexion were 53 shown to be negatively correlated with the maximum angle of ankle dorsiflexion (Miyamoto et 54 al., 2018). However, the findings of previous studies regarding the association between the lateral 55 gastrocnemius muscle stiffness assessed in a slightly lengthened position (i.e., at 0 ° of ankle 56 dorsiflexion) and maximum angle of ankle dorsiflexion are inconsistent (Miyamoto et al., 2018; 57 Reiner et al., 2024). Additionally, these studies did not evaluate nerve stiffness. Kawanishi et al. 58 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 22, 2025. ; https://doi.org/10.1101/2025.07.20.665743doi: bioRxiv preprint (2022) reported a negative correlation between the tibial nerve stiffness at 75% and 100% of the 59 maximum angle of ankle dorsiflexion and maximum angle of ankle dorsiflexion itself; however, 60 they did not evaluate muscle stiffness. These studies suggested that muscle and nerve stiffness 61 may be associated with the maximum angle of ankle dorsiflexion . However, few studies have 62 comprehensively investigated the association between both stiffness and the maximum angle of 63 ankle dorsiflexion. 64 Hirata et al. (2020) reported that in healthy young males, the triceps surae muscle 65 stiffness at 15° of ankle dorsiflexion was negatively correlated with the maximum angle of ankle 66 dorsiflexion, whereas the sciatic nerve stiffness was not. However, the tibial nerve, which may 67 affect the maximum angle of ankle dorsiflexion more strongly than the sciatic nerve, has not been 68 investigated. The tibial nerve stiffness near the ankle joint is negatively correlated with the 69 maximum angle of ankle dorsiflexion (Kawanishi et al., 2022) and is higher than the sciatic nerve 70 stiffness(Andrade et al., 2022) . Thus, it may reflect the stretch tolerance to ankle dorsiflexion 71 more strongly than the sciatic nerve stiffness. However, no study has investigated the association 72 between the triceps surae muscle and tibial nerve stiffness and maximum angle of ankle 73 dorsiflexion, and it is unclear which stiffness is associated most strongly. In addition, stiffness at 74 angles of ankle dorsiflexion larger than 15° may be more negatively associated with the maximum 75 angle of ankle dorsiflexion. 76 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 22, 2025. ; https://doi.org/10.1101/2025.07.20.665743doi: bioRxiv preprint The purpose of this study was to evaluate both triceps surae muscle and tibial nerve 77 stiffness and to clarify their association with the maximum angle of ankle dorsiflexion using shear 78 wave elastography. Negative correlations were found between ⅰ) the triceps surae muscle stiffness 79 and maximum angle of ankle dorsiflexion(Hirata et al., 2020; Miyamoto et al., 2018) and ⅱ) the 80 tibial nerve stiffness and maximum angle of ankle dorsiflexion (Kawanishi et al., 2022) . Based 81 on these correlations, we hypothesized that these muscles and nerve stiffness were associated with 82 the maximum angle of ankle dorsiflexion and that the association was stronger when the muscles 83 and nerve were greatly lengthened. 84 85 2. Methods 86 2.1. Participants 87 Forty-three healthy young adults who had no pain or reduced range of motions in their ankles on 88 the nondominant side participated in this study (19 males and 24 females, age 24.3 ± 2.9 years; 89 height 165.8 ± 7.7 cm; body mass 59.3 ± 8.2 kg). Thirty-nine of them were right-footed and four 90 were left-footed when the dominant foot was defined as the one that kicked a ball. An a priori 91 sample size estimation was performed using a linear multiple regression model in G*Power 92 (version 3.1.9.7; Heinrich Heine University, Düsseldorf, Germany) using an assumed effect size 93 of 0.35, an alpha level of 0.05, and a statistical power of 0.80. The analysis indicated that a 94 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 22, 2025. ; https://doi.org/10.1101/2025.07.20.665743doi: bioRxiv preprint minimum of 40 participants were required. Forty-three participants were recruited to account for 95 potentially missing data. The study protocols were explained to all the participants and informed 96 consent was obtained from each participant. This study conformed to the Declaration of Helsinki 97 and was approved by the Ethics Committee (approval number: C1652-2). 98 99 2.2. Experimental procedure 100 This cross-sectional study investigated the association between different shear wave velocit ies 101 and the maximum angle of ankle dorsiflexion. The shear wave velocities of the medial and lateral 102 gastrocnemius muscles, soleus muscle, and tibial nerve were measured. The participants were 103 placed in a prone position on the seat of a dynamometer (BIODEX System 4, BIODEX, NY , 104 USA) to measure the shear wave velocit ies and maximum angle . They were set at 0 ° of hip 105 flexion/extension, abduction/adduction, and internal/external rotation and 0° of knee 106 flexion/extension, with their feet attached to a plate and their feet and pelvis fixed using straps. 107 The participants were asked to relax during the measurements. To minimize the stretching effect, 108 first, the shear wave velocities were measured at 5°, 15°, and 25° of ankle dorsiflexion, in order, 109 and then the maximum angle of ankle dorsiflexion was measured. 110 111 2.3. Measurement of shear wave velocity 112 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 22, 2025. ; https://doi.org/10.1101/2025.07.20.665743doi: bioRxiv preprint The shear wave elastography mode in an ultrasound system (Aixplorer v12.2, SuperSonic 113 Imagine, Aix-en-Provence, France) with a linear probe ( 2–10 MHz, SuperLinear SL10-2) was 114 used to measure the shear wave velocity in each tissue. Each measurement was performed in a 115 musculoskeletal preset (muscle mode) (penetration mode, frequency: 1.7 Hz, smoothing level: 116 five, persistence high, opacity: 100%). Shear wave velocity (V) is used as an index of muscle and 117 nerve stiffness and is directly related to the shear modulus (G) as follows: 118 𝐺 = 𝜌𝑉2 119 where ρ is the estimated density of tissues (1.0 g/cm3) (Nordez et al., 2008). Muscle shear modulus 120 is strongly correlated with Young’s modulus, as assessed by traditional material testing (Eby et 121 al., 2013). However, the density of the tibial nerve could not be estimated as 1.0 g/cm 3, and we 122 adopted the shear wave velocity in this study. We interpreted that a higher shear wave velocity 123 indicates a higher stiffness of tissues. The shear wave velocities were measured at the following 124 locations: the medial and lateral gastrocnemius muscle s at 30% of the lower leg length (Akagi 125 and Takahashi, 2013; Nakamura et al., 2014), soleus muscle at 50% of the lower leg length (Kubo 126 et al., 2017), and tibial nerve at a point near the medial malleolus, as determined by preliminary 127 experiments. 128 129 2.4. Image analysis 130 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 22, 2025. ; https://doi.org/10.1101/2025.07.20.665743doi: bioRxiv preprint A region of interest (ROI) with a length of 1 cm and width of 2 cm was set. The Q -box trace 131 function was then used to surround the maximal area in the ROI, excluding the fascia, epineurium, 132 and artifacts, and the average shear wave velocity within the area was calculated. For each joint 133 angle, two ultrasound images of the triceps surae muscles and three ultrasound images of the tibial 134 nerve were acquired, and their mean values were used for statistical analysis. The analyzed 135 ultrasound images are shown in Fig. 1. 136 137 2.5. Measurement of maximum angle of ankle dorsiflexion 138 The ankle of a participant was passively dorsiflexed at 5°/s velocity from 30° of ankle plantar 139 flexion to the maximum angle at which the participant experienced discomfort without pain 140 (Nakamura et al., 2017). The participants were asked to press a button to stop the footplate upon 141 reaching the maximum angle of ankle dorsiflexion. The maximum angle was defined in 1-degree 142 increments. Before the measurement of the maximum angle of ankle dorsiflexion, two sessions 143 were conducted to familiarize the participants with the procedure (Hirata et al., 2015; Konrad et 144 al., 2015; Konrad and Tilp, 2014). The maximum angle of ankle dorsiflexion was measured three 145 times, and the mean value was used for statistical analysis. 146 147 2.6. Statistical analysis 148 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 22, 2025. ; https://doi.org/10.1101/2025.07.20.665743doi: bioRxiv preprint One participant who felt pain while measuring the shear wave velocit ies at 25 ° of ankle 149 dorsiflexion and another participant whose maximum angle of ankle dorsiflexion exceeded the 150 upper limit of the dynamometer were excluded. We performed statistical analysis of the data 151 obtained from 41 participants. SPSS Statistics 22 (IBM, Armonk, NY , USA) was used for the 152 statistical analysis. To confirm whether each tissue lengthened with increasing ankle dorsiflexion, 153 a multiple comparison test with Bonferroni correction was performed to compare the shear wave 154 velocities at 5°, 15°, and 25° of ankle dorsiflexion. To investigate the association between the 155 shear wave velocities of each tissue and maximum angle of ankle dorsiflexion, a multiple linear 156 regression analysis using the forced entry method was performed, specifying the shear wave 157 velocities of the four tissues as the independent variables and the maximum angle of ankle 158 dorsiflexion as a dependent variable. This multiple linear regression model was built at each angle 159 where the shear wave velocity was measured because previous studies have reported inconsistent 160 findings regarding the association between the shear wave velocity of a muscle in a slightly 161 lengthened position and maximum angle(Miyamoto et al., 2018; Reiner et al., 2024). Additionally, 162 to investigate the angle at which the shear wave velocity measured was most associated with the 163 maximum angle, a multiple linear regression analysis using the stepwise method was performed, 164 specifying the shear wave velocities that were significantly associated with the maximum angle 165 of ankle dorsiflexion in the forced entry method as the independent variables and the maximum 166 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 22, 2025. ; https://doi.org/10.1101/2025.07.20.665743doi: bioRxiv preprint angle of ankle dorsiflexion as a dependent variable. Statistical significance was set as p < 0.05. 167 168 3. Results 169 3.1. Characteristics and shear wave velocity of participants 170 The characteristics of the 41 participants included in the analysis were shown as follows; 19 males 171 and 22 females, 37 right-footed and 4 left-footed, age—24.3 ± 2.9 years; height—166.2 ± 7.6 cm; 172 body mass—59.8 ± 7.6 kg. The mean ± SD of their maximum angle of ankle dorsiflexion was 32 173 ± 7°. For the medial and lateral gastrocnemius muscles, the shear wave velocities at 15° of ankle 174 dorsiflexion were significantly higher than those at 5° of ankle dorsiflexion, and the shear wave 175 velocities at 25° of ankle dorsiflexion were significantly higher than those at 5° and 15° of ankle 176 dorsiflexion (p < 0.001 for all the angles). For the soleus muscle, no significant difference was 177 found in the shear wave velocit ies at 5° and 15° of ankle dorsiflexion ( p = 0.557), whereas the 178 shear wave velocity at 25° of ankle dorsiflexion was significantly higher than those at 5° and 15° 179 of ankle dorsiflexion (both p < 0.001). For the tibial nerve, the shear wave velocity at 15° of ankle 180 dorsiflexion was significantly higher than that at 5° of ankle dorsiflexion ( p = 0.001), and the 181 shear wave velocity at 25° of ankle dorsiflexion was significantly higher than those at 5° (p < 182 0.001) and 15° (p = 0.002) of ankle dorsiflexion. The shear wave velocity of each tissue at each 183 angle is listed in Table 1. 184 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 22, 2025. ; https://doi.org/10.1101/2025.07.20.665743doi: bioRxiv preprint 185 3.2. Multiple linear regression analysis of shear wave velocity and maximum angle of ankle 186 dorsiflexion 187 The results of the multiple linear regression analysis using the forced entry method are presented 188 in Table 2. The shear wave velocities of the tibial nerve at 5° (adjusted R2 = 0.045), 15° (adjusted 189 R2 = 0.177), and 25° (adjusted R2 = 0.138) of ankle dorsiflexion were significantly and negatively 190 associated with the maximum angle of ankle dorsiflexion. However, the shear wave velocities of 191 the triceps surae muscles at 5°, 15°, and 25° of ankle dorsiflexion were not significantly associated 192 with the maximum angle of ankle dorsiflexion . Multiple linear regression analysis was also 193 conducted using the stepwise method, specifying the shear wave velocities of the tibial nerve at 194 5°, 15°, and 25° of ankle dorsiflexion as the independent variables. Its results showed that only 195 the shear wave velocity of the tibial nerve at 25° of ankle dorsiflexion was significantly and 196 negatively associated with the maximum angle of ankle dorsiflexion ( unstandardized regression 197 coefficient = −2.618, standardized regression coefficient = −0.449, 95% confidence interval = 198 from −4.305 to −0.931, p = 0.003, adjusted R2 = 0.181). 199 200 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 22, 2025. ; https://doi.org/10.1101/2025.07.20.665743doi: bioRxiv preprint 201 4. Discussion 202 In this study, we investigated the association between the shear wave velocit ies of the triceps 203 surae muscles and tibial nerve and the maximum angle of ankle dorsiflexion. We found that the 204 shear wave velocities of the tibial nerve at 5°, 15°, and 25° of ankle dorsiflexion were negatively 205 associated with the maximum angle and that the shear wave velocity of the tibial nerve at 25° of 206 ankle dorsiflexion was the most negatively associated. However, the shear wave velocities of the 207 triceps surae muscles measured at these angles were not associated with the maximum angle. This 208 is the first study to show that the tibial nerve stiffness is more strongly associated with the 209 maximum angle of ankle dorsiflexion than the triceps surae muscle stiffness. 210 The significant association between the shear wave velocity of the tibial nerve and 211 maximum angle of ankle dorsiflexion is aligned with our hypothesis and the findings of a previous 212 study(Kawanishi et al., 2022). Andrade et al. (2018) suggested that nerve stiffness reflects stretch 213 tolerance and that its potential mechanism is an intrinsic nerve such as the nervi nervorum. The 214 nervi nervorum may be related to pain threshold (Bove and Light, 1995; Marshall, 1883) and is 215 sensitive to stretching along the long axis of the nerve (Andrade et al., 2018) . Individuals with 216 high tibial nerve stiffness may have poor stretch tolerance to ankle dorsiflexion owing to a 217 sensitive nervi nervorum. However, the mechanism underlying the effect of nerve stiffness on 218 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 22, 2025. ; https://doi.org/10.1101/2025.07.20.665743doi: bioRxiv preprint stretch tolerance remains unclear. Additionally, using the stepwise method, we found that only the 219 shear wave velocity of the tibial nerve at 25 ° of ankle dorsiflexion was associated with the 220 maximum angle of ankle dorsiflexion. The tibial nerve stiffness in a greatly lengthened position 221 may be more strongly associated with the maximum angle of ankle dorsiflexion than that in a 222 slightly lengthened position. The greatly lengthened nervi nervorum of the tibial nerve may reflect 223 stretch tolerance to ankle dorsiflexion, although the underlying mechanism has not been clarified. 224 The nonsignificant association between the shear wave velocit ies of the triceps surae 225 muscles and maximum angle of ankle dorsiflexion was not aligned with our hypothesis and 226 previous studies (Hirata et al., 2020; Miyamoto et al., 2018) . Conventionally, both muscle 227 stiffness and stretch tolerance are considered to be associated with the maximum joint angle 228 (Magnusson et al., 1997). Previous studies have reported a nonsignificant association between the 229 maximum angle of ankle dorsiflexion and medial and lateral gastrocnemius muscle stiffness in a 230 slightly lengthened position (Reiner et al., 2024) and a significant association between them in a 231 greatly lengthened position (Hirata et al., 2020; Miyamoto et al., 2018) . However, in this study, 232 the shear wave velocities of the triceps surae muscles, even in greatly lengthened positions, were 233 not associated with the maximum angle of ankle dorsiflexion . This study suggested that stretch 234 tolerance affects the maximum joint angle more strongly than muscle stiffness. Nakamura et al. 235 (2021) reported that an increase in the maximum angle of ankle dorsiflexion did not correlate with 236 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 22, 2025. ; https://doi.org/10.1101/2025.07.20.665743doi: bioRxiv preprint a decrease in the medial gastrocnemius muscle stiffness after static stretching intervention. 237 Although this was a cross-sectional study and not an intervention study, muscle stiffness may be 238 less strongly associated with the maximum joint angle than stretch tolerance. 239 This study had some limitations. First, the participants were healthy young adults. Hirata 240 et al. (2020) reported that tissue stiffness correlat ion with the maximum joint angle differed 241 between young and elderly people. Additionally, because the gastrocnemius muscle stiffness of 242 stroke patients is higher than that of healthy individuals (Belghith et al., 2024; Le Sant et al., 243 2019), in such patients, muscle stiffness may be associated with the maximum joint angle. Second, 244 the shear wave velocities of the triceps surae muscles and tibial nerve were measured at a single 245 point. Previous studies reported regional difference s in the medial and lateral gastrocnemius 246 muscles (Le Sant et al., 2017; Zhou et al., 2019) and tibial nerve stiffness (Andrade et al., 2022). 247 A region where the tissue stiffness is more strongly associated with the maximum joint angle may 248 be found by measuring the shear wave velocities at other points. Third, we measured the shear 249 wave velocities and maximum angles with only 0° of knee flexion. Previous studies have reported 250 that when the ankle moves from plantar flexion to dorsiflexion, the change in the triceps surae 251 muscle stiffness differs for 0° and 90° of knee flexion (Ateş et al., 2018; Le Sant et al., 2017). The 252 maximum angle of ankle dorsiflexion with different angle of knee flexion may be associated with 253 tissue stiffness other than that of the tibial nerve. 254 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 22, 2025. ; https://doi.org/10.1101/2025.07.20.665743doi: bioRxiv preprint Consequently, the tibial nerve stiffness was negatively associated with the maximum 255 angle of ankle dorsiflexion , whereas the triceps surae muscle stiffness was not. Because nerve 256 stiffness is considered to reflect stretch tolerance (Andrade et al., 2018), stretch tolerance may be 257 more strongly associated with the maximum angle of ankle dorsiflexion than muscle stiffness. 258 Additionally, the tibial nerve stiffness in a greatly lengthened position was more strongly 259 associated with the maximum angle of ankle dorsiflexion than in a slightly lengthened position. 260 Nerve stiffness in a greatly lengthened position may more strongly reflect stretch tolerance. 261 262 Declaration of Ethics 263 All relevant ethical guidelines have been followed, all necessary ethics committee approvals have 264 been obtained, all necessary participant consent has been obtained, and the appropriate 265 institutional forms have been archived. 266 267 Declaration of Competing Interest 268 The authors declare that they have no known competing financial interests or personal 269 relationships that could have appeared to influence the work reported in this paper. 270 271 Funding 272 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 22, 2025. ; https://doi.org/10.1101/2025.07.20.665743doi: bioRxiv preprint This work was supported by JST SPRING (grant number JPMJSP2110). 273 274

275 We are grateful to Editage (www.editage.jp) for assistance with English language editing. 276 277 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 22, 2025. ; https://doi.org/10.1101/2025.07.20.665743doi: bioRxiv preprint

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No reuse allowed without permission. The copyright holder for this preprintthis version posted July 22, 2025. ; https://doi.org/10.1101/2025.07.20.665743doi: bioRxiv preprint Gastrocnemius Passive Properties. Front Physiol 12. https://doi.org/10.3389/fphys.2021.656579 349 Nordez, A., Gennisson, J.L., Casari, P., Catheline, S., Cornu, C., 2008. Characterization of muscle 350 belly elastic properties during passive stretching using transient elastography. J Biomech 41, 351 2305–2311. https://doi.org/10.1016/j.jbiomech.2008.03.033 352 Reiner, M.M., Tilp, M., Nakamura, M., Konrad, A., 2024. Is muscle stiffness a determinant for range 353 of motion in the leg muscles? Biol Sport 41, 115 –121. 354 https://doi.org/10.5114/biolsport.2024.131821 355 Zhou, J., Liu, C., Zhang, Z., 2019. Non -uniform stiffness within gastrocnemius -achilles tendon 356 complex observed after static stretching. J Sports Sci Med 18, 454–461. 357 358 359 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 22, 2025. ; https://doi.org/10.1101/2025.07.20.665743doi: bioRxiv preprint Figure and figure legend 360 361 Fig. 1: The analyzed ultrasound images. a: medial gastrocnemius muscle, b: lateral gastrocnemius 362 muscle, c: soleus muscle, d: tibial nerve. The areas surrounded by white dashed lines are analyzed. 363 364 Tables and table legends 365 Table 1: Shear wave velocity of each tissue at each angle. 366 Shear wave velocity (m/s) 5° of ankle dorsiflexion 15° of ankle dorsiflexion 25° of ankle dorsiflexion Medial gastrocnemius muscle 4.59 ± 0.61 6.45 ± 0.78* 8.32 ± 0.82*§ Lateral gastrocnemius muscle 4.05 ± 0.66 5.25 ± 0.67* 6.95 ± 0.88*§ Soleus muscle 3.31 ± 0.90 3.46 ± 0.75 4.00 ± 0.93*§ Tibial nerve 6.74 ± 1.31 7.24 ± 1.40* 7.78 ± 1.23*§ Shear wave velocit ies are described as mean ± SD. *: significantly higher than 5° of ankle 367 dorsiflexion (p < 0.05), §: significantly higher than 15° of ankle dorsiflexion (p < 0.05). 368 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 22, 2025. ; https://doi.org/10.1101/2025.07.20.665743doi: bioRxiv preprint 369 Table 2: The results of multiple regression analysis. 370 5° of ankle dorsiflexion B β 95%CI p value Medial gastrocnemius muscle 0.955 0.081 -3.362 to 5.272 0.656 Lateral gastrocnemius muscle -0.939 -0.086 -5.117 to 3.298 0.656 Soleus muscle 0.054 0.007 -2.715 to 2.822 0.969 Tibial nerve -1.977 -0.361 -3.759 to -0.195 0.031* 15° of ankle dorsiflexion B β 95%CI p value Medial gastrocnemius muscle -3.174 -0.345 -6.908 to 0.559 0.093 Lateral gastrocnemius muscle 3.728 0.347 -1.194 to 8.650 0.133 Soleus muscle 1.355 0.141 -1.690 to 4.399 0.373 Tibial nerve -2.954 -0.575 -4.719 to -1.188 0.002* 25° of ankle dorsiflexion B β 95%CI p value Medial gastrocnemius muscle -0.808 -0.092 -3.686 to 2.071 0.573 Lateral gastrocnemius muscle 1.101 0.134 -1.671 to 3.874 0.426 Soleus muscle 0.582 0.075 -1.844 to 3.008 0.630 Tibial nerve -2.880 -0.494 -4.702 to -1.059 0.003* *: p < 0.05, B: unstandardized regression coefficient, β: standardized regression coefficient, CI: 371 confidence interval. 372 373 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 22, 2025. ; https://doi.org/10.1101/2025.07.20.665743doi: bioRxiv preprint