Hamstring muscle strain injury is one of the most commonly seen injuries in sports such as track and field, soccer, football, and rugby. The purpose of this study was to advance our understanding of the mechanisms of hamstring muscle strain injuries during over ground sprinting by investigating hamstring muscle-tendon kinematics and muscle activation. Three-dimensional videographic and electromyographic (EMG) data were collected for 20 male runners, soccer or lacrosse players performing overground sprinting at their maximum effort. Hamstring muscle-tendon lengths, elongation velocities, and linear envelop EMG data were analyzed for a running gait cycle of the dominant leg. Hamstring muscles exhibited eccentric contractions during the late stance phase as well as during the late swing phase of overground sprinting. The peak eccentric contraction speeds of the hamstring muscles were significantly greater during the late swing phase than during the late stance phase (p=0.001) while the hamstring muscle-tendon lengths at the peak eccentric contraction speeds were significantly greater during the late stance phase than during the late swing phase (p=0.001). No significant differences existed in the maximum hamstring muscle-tendon lengths between the two eccentric contractions. The potential for hamstring muscle strain injury exists during the late stance phase as well as during the late swing phases of overground sprinting.