The purpose of the present study was to investigate the effect of varying inter-contraction intervals on central and peripheral muscle fatigue during intermittent contractions. Six healthy men carried out maximal unilateral isometric plantar flexions 50 times, separated with an interval of 2, 4, 10, or 30 s. Supramaximal electrical stimuli (twitches) were imposed percutaneously on the tibial nerve during and after every 10th contraction to assess the level of voluntary activation. The surface electromyogram (EMG) was recorded from the medial and lateral gastrocnemius (MG and LG) and soleus (Sol) muscles. Plantar flexion torque and other parameters were maintained over contractions with 30-s intervals, while the torque as well as EMG activity of the MG, LG and Sol and the level of voluntary activation decreased significantly under conditions using 2-, 4-, and 10-s intervals. The amount of decrease in the parameters was greater for shorter intervals. With 2-s intervals, the twitch torque decreased significantly, the half-relaxation time of the twitch torque increased significantly, and the EMG mean power frequency of the MG and LG shifted significantly toward lower frequencies, whereas no significant changes were found under other conditions. These results indicate that there are differences in the contributions of central and peripheral fatigue, both of which are a function of inter-contraction interval.