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Hamstring injury has been associated with fatigue-induced reductions in activation levels during running. This study examined neuromuscular changes of the hamstring muscles as a result of fatigue following sprinting in a group of nine team sport athletes. Hamstring muscle activation, lower-limb kinematics and isokinetic eccentric hamstring strength were assessed to examine the effects of fatigue during running at five different sub-maximal speeds. As expected, there were significant increases in both Biceps Femoris (BF) and Semitendinosus (ST) activations with running speed (P < 0.001). After fatigue, BF activation during late swing significantly decreased by an average of 11% (P=0.002). There was evidence in some subjects that ST activity was increased with fatigue but the increase (4%) was non-significant for the group. There was also a tendency for reduced BF activity with fatigue to be more evident at the faster speeds of running. These findings support other evidence in the literature that the lateral hamstrings (BF) are more susceptible to fatigue. In addition, there were signs of compensatory increased ST activation levels in some subjects. These effects lend support to the potential benefit of this neuromuscular assessment of the hamstrings as a useful measure of both performance and recovery.