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Purpose: Hamstring muscle strain injury is very common in sports involving high-speed running. Hamstring muscles originate from the ischial tuberosity and thus pelvic position may influence hamstring strain during different sports movements like sprinting, but this has only been evaluated by indirect methods. This study tested the hypothesis that a change in anterior pelvic tilt causes elongation of the overall hamstring complex and disproportionately elongates proximal relative to distal muscle regions., Methods: Seven fresh-frozen specimens (full lower limb with pelvis and lumbar spine) were used for this in vitro study. Specimens were dissected to enable visualization of the hamstring muscles and then fixed into a custom-made testing bench that allowed controlled movement of the pelvis over a fixed femur and tibia. Nine markers were inserted into the hamstring muscles to allow intra- and intermuscle difference measurements. Then, six different anterior pelvic angles were used to measure the difference in hamstring muscle lengthening through a three-dimensional reconstruction system based on stereoscopic machine vision technology., Results: An increase in anterior pelvic tilt produced a significant non-uniform increase in tissue elongation in all regions of the three hamstring muscles (semitendinosus, semimembranosus [SMB] and biceps femoris long head), which was greater in the proximal (>1 cm every 5°) compared to the distal region (≈0.4 cm every 5°). At the proximal hamstring region, SMB showed significantly greater length changes compared to conjoint tendons with nonstatistically significant elongation differences between muscles at the distal region., Conclusion: Considering the results of the study, the pelvis segment will likely play a fundamental role as a strain regulator of hamstring muscles. These results will have an impact on injury rehabilitation and prevention processes of hamstring injuries, as well as optimize future musculoskeletal models and avoid potential underestimation of the hamstring muscle-tendon complex lengthening during high-speed running., Level of Evidence: N/A., (© 2024 European Society of Sports Traumatology, Knee Surgery and Arthroscopy.)