The primary aim of this study was to examine central fatigue of the plantar flexor muscle group after prolonged running using the twitch interpolation technique. Eight healthy, habitually active male subjects ran on a motorized treadmill for 2 h at a speed corresponding to 75% of peak oxygen uptake (VO(2peak)). Maximal voluntary isometric contraction (MVC) strength as well as the electrically induced twitch produced during MVC [interpolated twitch (IT)] and at rest [resting twitch (RT)] were measured before and after running. The level of activation (LOA) during each MVC was calculated as LOA (%)=100(1-IT/RT). Both MVC and LOA decreased (17+/-16% and 19+/-15%, respectively, P<0.05) after running, whereas RT did not change. The decrease in MVC was correlated with the decrease in LOA (r=0.87, P<0.05). The results demonstrate that after 2 h of treadmill running at an intensity of 75% of VO(2peak), there was a reduction in maximal voluntary plantar flexor muscle strength that was mainly related to central fatigue.