A thorough understanding of the biomechanics of the hamstrings during sprinting is required to optimise injury rehabilitation and prevention strategies. The main aims of this study were to compare hamstrings load across different modes of locomotion as well as before and after an acute sprinting-related muscle strain injury. Bilateral kinematic and ground reaction force data were captured from a single subject whilst walking, jogging and sprinting prior to and immediately following a significant injury involving the right semitendinosis and biceps femoris long head muscles. Experimental data were input into a three-dimensional musculoskeletal model of the body and used, together with optimisation theory, to determine lower-limb muscle forces for each locomotor task. Hamstrings load was found to be greatest during terminal swing for sprinting. The hamstrings contributed the majority of the terminal swing hip extension and knee flexion torques, whilst gluteus maximus contributed most of the stance phase hip extension torque. Gastrocnemius contributed little to the terminal swing knee flexion torque. Peak hamstrings force was also substantially greater during terminal swing compared to stance for sprinting, but not for walking and jogging. Immediately following the muscle strain injury, the hamstrings demonstrated an intolerance to perform an eccentric-type contraction. Whilst peak hamstrings force during terminal swing did not decrease post-injury, both peak hamstrings length and negative work during terminal swing were considerably reduced. These results lend support to the paradigm that the hamstrings are most susceptible to muscle strain injury during the terminal swing phase of sprinting when they are contracting eccentrically.