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The hamstring plays an important role in reducing loads born by the anterior cruciate ligament. As anterior cruciate ligament injuries occur rapidly after ground contact, how the hamstring is activated prior to landing can influence injury risk. The purpose was to determine sex-related differences in hamstring activation immediately before landing and the effect of fatigue on "preactivation." Twenty-four participants (13 males and 11 females, age = 24.3 [6.5] y, mass = 72.2 [19.3] kg, height = 169 [9.7] cm) participated in this study. Participants completed a drop-vertical jump protocol before and after a lower body fatigue protocol. Hamstring electromyography (EMG) amplitude at 5 periods prior to landing, peak vertical ground reactions forces (in newtons/body weight), rate of loading (in body weight/second), and landing error scoring system were measured. Females had higher EMG amplitude before and after fatigue (P <.024), with decreased EMG amplitude for both sexes after fatigue (P =.025). There was no change on vertical ground reaction force, rate of loading, or landing error scoring system. Males and females demonstrated similar landing performance before and after fatigue but have different hamstring neuromuscular coordination strategies. The acute reduction in hamstring EMG amplitude following fatigue may increase loading on the anterior cruciate ligament. [ABSTRACT FROM AUTHOR]