Skeletal muscle thickness is a valuable indicator of several aspects of a muscle's functional capabilities. We used computational analysis of ultrasound images, recorded from 10 humans walking and running at a range of speeds (0.7-5.0 m s ), to quantify interactions in thickness change between three ankle plantar flexor muscles (soleus, medial and lateral gastrocnemius) and quantify thickness changes at multiple muscle sites within each image. Statistical analysis of thickness change as a function of stride cycle (1d statistical parametric mapping) revealed significant differences between soleus and both gastrocnemii across the whole stride cycle as they bulged within the shared anatomical space. Within each muscle, changes in thickness differed between measurement sites but not locomotor condition. For some of the stride, thickness measures taken from the distal-mid image region represented the mean muscle thickness, which may therefore be a reliable region for these measures. Assumptions that muscle thickness is constant during a task, often made in musculoskeletal models, do not hold for the muscles and locomotor conditions studied here and researchers should not assume that a single thickness measure, from one point of the stride cycle or a static image, represents muscle thickness during dynamic movements.