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The hamstring muscles are well positioned to limit both anterior tibial translation and anterior cruciate ligament strain during the knee flexion phase of a jump landing. We hypothesized that systematically increasing or decreasing hamstring tension during the knee flexion phase of a simulated jump landing would significantly affect peak relative strain in the anterior cruciate ligament.Ten cadaveric knees from four male and six female donors (mean age [and standard deviation] at the time of death, 60.3 ± 23.6 years) were mounted in a custom fixture to initially position the specimen in 25° of knee flexion and simulate axial impulsive loading averaging 1700 N to cause an increase in knee flexion. Quadriceps, hamstring, and gastrocnemius muscle forces were simulated with use of pretensioned linear springs, with the tension in the hamstrings arranged to be increased, held constant, decreased, at “baseline,” or absent during knee flexion. Impulsive loading applied along the tibia and femur was monitored with use of triaxial load transducers, while uniaxial load cells monitored quadriceps and medial and lateral hamstring forces. Relative strain in the anterior cruciate ligament was measured with use of a differential variable reluctance transducer, and tibiofemoral kinematics were measured optoelectronically. For each specimen, anterior cruciate ligament strains were recorded over eighty impact trials: ten preconditioning trials, ten “baseline” trials involving decreasing hamstring tension performed before and after three sets of ten trials conducted with increasing hamstring tension, constant hamstring tension, or no hamstring tension. Peak relative strains in the anterior cruciate ligament were normalized for comparison across specimens.Increasing hamstring force during the knee flexion landing phase decreased the peak relative strain in the anterior cruciate ligament by >70% compared with the baseline condition (p = 0.005). Neither a constant hamstring muscle force nor the absence of a hamstring force significantly changed the peak strain in the anterior cruciate ligament relative to the baseline condition.Increasing hamstring muscle force during the knee flexion phase of a simulated jump landing significantly reduces the peak relative strain in the anterior cruciate ligament in vitro.Clinical Relevance: It may be possible to proactively limit peak anterior cruciate ligament strain during the knee flexion phase of jump landings by accentuating hip flexion, thereby increasing the tension in active hamstring muscles by lengthening them.