Background. The relationship between hamstring flexibility and the risk of OSD continues to be a debate, and whether hamstring stretching exercises should be considered as one of the conservative treatments of OSD is still unclear. Objectives. To investigate the relationship between hamstring flexibility and the risk of OSD by assessing the changes of loading on the tibial tuberosity caused by the changes of hamstring optimal lengths. Methods. Experimental data of a young adult running at 4 m/s were used, which were collected by an eight-camera motion capture system together with an instrumented treadmill. Muscle forces were estimated in OpenSim when hamstring optimal lengths changed in the range of 70–130% of the control case in 5% increments. The force and accumulated force of quadriceps muscle were calculated to evaluate the impact of hamstring optimal lengths on the loading on tibial tuberosity. The changes in muscle forces throughout the gait cycle were compared by using statistical parametric mapping (SPM). The average peak force and accumulated force of five gait cycles were compared. Results. Although the maximum force of the quadriceps muscle was slightly affected by changes in hamstring optimal lengths, the accumulated force of quadriceps muscle increased by 21.97% with hamstring optimal lengths decreased by 30% of the control case. The increase of the muscle force mainly occurred in the early stance phase and terminal swing phase ( P < 0.05 ). However, when hamstring optimal lengths were longer than the control, it had a little effect on accumulated force of quadriceps muscle. Conclusions. The results of this study indicate that a shorter hamstring optimal length, which means lack of flexibility, can cause a high accumulated force on tibial tuberosity, thus increasing the risk of OSD. Hamstring stretching exercise is only effective for people with lack of hamstring flexibility.