The effects of short-term high-intensity exercise on single fiber contractile function in humans are unknown. Therefore, the purposes of this study were: (a) to access the acute effects of repeated high-intensity exercise on human single muscle fiber contractile function; and (b) to examine whether contractile function was affected by alterations in the redox balance. Eleven elite cross-country skiers performed four maximal bouts of 1300m treadmill skiing with 45min recovery. Contractile function of chemically skinned single fibers from triceps brachii was examined before the first and following the fourth sprint with respect to Ca2+ sensitivity and maximal Ca2+-activated force. To investigate the oxidative effects of exercise on single fiber contractile function, a subset of fibers was incubated with dithiothreitol (DTT) before analysis. Ca2+ sensitivity was enhanced by exercise in both MHC I (17%, P<0.05) and MHC II (15%, P<0.05) fibers. This potentiation was not present after incubation of fibers with DTT. Specific force of both MHC I and MHC II fibers was unaffected by exercise. In conclusion, repeated high-intensity exercise increased Ca2+ sensitivity in both MHC I and MHC II fibers. This effect was not observed in a reducing environment indicative of an exercise-induced oxidation of the human contractile apparatus.