We aimed to clarify whether post-activation potentiation (PAP) is associated with the amount of fascicle shortening of individual muscles during twitch and whether this relationship depends on muscle fiber composition in humans. Eighteen healthy young adults (four female) participated in this study. Single supramaximal electrical stimulations were applied to the tibial nerve to elicit plantarflexion twitch, involving the medial gastrocnemius (MG) with approximately 50 % Type I fibers and the synergist soleus (SOL) with more than 80 % Type I fibers. The stimuli were delivered before (Pre), immediately after (Post-0 min), 5 min after (Post-5 min), and 10 min after a 6-s maximal voluntary isometric plantarflexion contraction (MVC) and peak torque (PT) during twitch contraction were calculated. The instantaneous fascicle length of each muscle was measured using ultrasound B-mode images acquired at 125 fps during twitch contraction and the amount of fascicle shortening (ΔFL) was calculated. PT was greater after MVC than that at Pre (P < 0.05). The ΔFL of both MG and SOL were greater at Post-0 min and Post-5 min than at Pre (P < 0.05). PT and ΔFL at Post-0 min relative to values at Pre were positively correlated in the MG (r = 0.624, P = 0.006), but not in the SOL. These results suggest that the contribution to PAP of isometric plantarflexion is greater from the MG than that from the SOL, implying a dependence of PAP on muscle fiber composition.