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The purpose of the current study was to examine the reproducibility of fascicle length and pennation angle of gastrocnemius medialis while human walking. To the best of our knowledge, this is the first study of the reproducibility of fascicle length and pennation angle of gastrocnemius medialis in vivo during human gait. Twelve males performed 10 gait trials on a treadmill, in 2 separate days. B-mode ultrasonography, with the ultrasound probe firmly adjusted in the transverse and frontal planes using a special cast, was used to measure the fascicle length and the pennation angle of the gastrocnemius medialis (GM). A Vicon 624 system with three cameras operating at 120 Hz was also used to record the ankle and knee joint angles. The results showed that measurements of fascicle length and pennation angle showed high reproducibility during the gait cycle, both within the same day and between different days. Moreover, the root mean square differences between the repeated waveforms of both variables were very small, compared with their ranges (fascicle length: RMS = ∼3 mm, range: 38–63 mm; pennation angle: RMS = ∼1.5°, range: 22–32°). However, their reproducibility was lower compared to the joint angles. It was found that representative data have to be derived by a wide number of gait trials (fascicle length ∼six trials, pennation angle more than 10 trials), to assure the reliability of the fascicle length and pennation angle in human gait.