The neural activation (iEMG) and selected stride characteristics of six male sprinters were studied for 100-, 200-, 300- and 400-m experimental sprints, which were run according to the velocity in the 400 m. Blood lactate (BLa) was analysed and drop jumps were performed with EMG registration at rest and after each sprint. Running velocity (P less than 0.001) and stride length (P less than 0.05) decreased and contact time increased (P less than 0.01) during the 400-m sprint. The increase in contact time was greatest immediately after runs of 100 and 300 m. The peak BLa increased and the rate of BLa accumulation decreased with running distance (P less than 0.001). The height of rise of the centre of mass in the drop jumps was smaller immediately after the 300 m (P less than 0.05) and the 400 m (P less than 0.01) than at rest, and it correlated negatively with peak BLa (r = -0.77, P less than 0.001). The EMG and EMG:running velocity ratio increased with running distance. It was concluded that force generation of the leg muscles had already begun to decrease during the first quarter of the 400-m sprint. The deteriorating force production was compensated for until about 200-300 m. Thereafter, it was impossible to compensate for fatigue and the speed of running dropped. According to this study, fatigue in the 400-m sprint among trained athletes is mainly due to processes within skeletal muscle rather than the central nervous system.