OBJECTIVES:To investigate if the temporal characteristics of hamstring and gluteal muscle activation are altered during high speed overground running in professional Australian Football players following hamstring muscle injury. DESIGN:Cohort study. SETTING:Field-based testing. PARTICIPANTS:Elite professional Australian Football players who had sustained a hamstring muscle injury in the six months prior to testing (n = 7) and a group of players from the same club who had no history of hamstring muscle injury (n = 8). MAIN OUTCOME MEASURES:Muscle onset timing, muscle offset timing and muscle onset duration of the medial hamstrings, biceps femoris and gluteus maximus muscles during high-speed running using electromyographic data. RESULTS:No significant differences in any of the temporal aspects of muscle activation were found between groups for any of the muscles tested (p > 0.05). CONCLUSIONS:Persistent alterations to the timing of muscle activation following hamstring muscle injury that have been reported in recreational athletes were not observed during high speed running in professional athletes who have completed comprehensive rehabilitation programs.