Strengthening of the hamstrings is often recommended following injury and reconstruction of the anterior cruciate ligament. It has been suggested that hamstrings activity stabilizes the knee and reduces anterior cruciate ligament load during weight-bearing flexion; however, the effects of hamstrings cocontraction on the kinematics and mechanics of the normal knee have not been assessed at physiological load levels. The aim of this study was to determine whether the addition of hamstrings force affects knee rotations, translations, and joint and quadriceps force during flexion with loads at physiological levels applied to the muscles and joints. Eight cadaveric knee specimens were tested with a servohydraulic mechanism capable of applying controlled dynamic loads to simulate quadriceps and hamstrings muscle forces throughout a physiological range of motion. A constant vertical load of physiologic magnitude was applied to the hip, and quadriceps force was varied to maintain equilibrium throughout flexion. Two conditions were tested: no hamstrings force and a constant hamstrings force equivalent to the vertical load. Hamstrings force significantly reduced internal rotation (p<0.0001) and anterior translation (p<0.0001), increased quadriceps force (p<0.0001) and normal resultant force on the tibia (p<0.0001), and reversed the direction of the shear force on the tibia (p<0.0001). These results suggest that hamstrings strengthening following anterior cruciate ligament injury may benefit anterior cruciate ligament-deficient and reconstructed knees by reducing the load in the ligament; however, they also imply that this comes at the expense of efficiency and higher patellofemoral and joint forces.