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Purpose Sprinting is the most common activity associated with hamstring strain injuries in soccer. However, the potential mechanisms for hamstring strain injuries during the initial acceleration phase of sprinting are unclear. Therefore, this study aimed to characterize the kinematics and activation of the hamstring muscles in soccer players to define the potential mechanism of hamstring strain injury.Methods Ten male soccer players with no lower-limb injuries in the past year were recruited for this study. Each participant was instructed to perform a minimum of three successful 15 m sprints. Hamstring kinematics and activations were estimated by employing musculoskeletal modeling and wireless electromyography to define potential injury mechanisms.Results During the initial acceleration phase, the hamstring muscles undergo great strain and high activation levels during the late swing and early stance phases. The hamstring muscles experienced great strain and overload during the late swing and foot strike of the running gait cycle. Additionally, the biceps femoris long head was significantly more stretched than the semimembranosus and semitendinosus (p < 0.0001, p < 0.05).Conclusion Based on these results, hamstring injuries are likely to occur during the late-swing and early stance of the running gait cycle, with the biceps femoris long head being more susceptible to injuries than other hamstring muscles.