This study aimed to clarify the influence of temperature on post-activation twitch potentiation, a possible indicator of fast-twitch fiber activation during a preceding voluntary contraction. Ten healthy males immersed their left lower leg in water of different temperatures (cold: 0°C, neutral: 32-35°C, hot: ~43°C) for 20 min each. In each temperature condition, they performed submaximal (10%-50% of maximal voluntary contraction torque measured before water immersion) and maximal plantar flexions. Immediately after each voluntary contraction, twitch contractions were evoked with supramaximal stimulation of the posterior tibial nerve. The magnitude of twitch potentiation, defined as a percent increase in twitch torque following a voluntary contraction, increased with the intensity of the preceding voluntary contraction. The magnitude of twitch potentiation after the maximal voluntary contraction was smaller in Cold than in the other temperature conditions. However, temperature had no influence on the relative magnitude of twitch potentiation following the submaximal contractions. In addition, there was no difference in electromyographic activity between the gastrocnemius and soleus muscles in any temperature conditions. Collectively, the temperature dependence was not observed when using twitch potentiation or electromyographic amplitude as an indicator of fast-twitch fiber activation during brief submaximal voluntary contractions.