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The aim of our study was to investigate the effect of muscle-specific fatigue of the quadriceps and hamstring muscles on the biomechanical factors of anterior cruciate ligament (ACL) injury using musculoskeletal modeling techniques during directional diversion maneuver. Fifteen female subjects performed a directional diversion maneuver under three treatment conditions (quadriceps fatigue, hamstring fatigue, and control gait). Data from the 3D motion capture system and force platform were used to extract anterior/posterior ACL forces using the two-bundle ACL musculoskeletal modeling approach. A decrease in maximum extension (51.3%) and flexion (50.7%) torque after fatigue was observed. After quadriceps fatigue, the extension (p = 0.041) and adduction moments (p = 0.046) of the knee joint and the mean anterior bundle of ACL force (p = 0.021) decreased significantly. The knee flexion angle (p = 0.003), knee valgus angle (p = 0.013), and shear force (p = 0.043) decreased significantly after hamstring fatigue. The decrease in ACL force after quadriceps fatigue confirms its significant role in causing an ACL injury. However, no significant differences in ACL load after hamstring fatigue leads us to speculate that the antagonist muscle group, i.e., the hamstring, might not have a preventive mechanism against ACL injury.