A change in hamstring strength and activation is typically seen after injuries or invasive surgeries such as anterior cruciate reconstruction or total knee replacement. While many studies have investigated the influence of isometric increases in hamstring load on knee joint kinematics, few have quantified the change in kinematics due to a variation in medial to lateral hamstring force ratio. This study examined the changes in knee joint kinematics on eight cadaveric knees during an open-chain deep knee bend for six different loading configurations: five loaded hamstring configurations that varied the ratio of a total load of 175 N between the semimembranosus and biceps femoris and one with no loads on the hamstring. The anterior-posterior translation of the medial and lateral femoral condyles' lowest points along proximal-distal axis of the tibia, the axial rotation of the tibia, and the quadriceps load were measured at each flexion angle. Unloading the hamstring shifted the medial and lateral lowest points posteriorly and increased tibial internal rotation. The influence of unloading hamstrings on quadriceps load was small in early flexion and increased with knee flexion. The loading configuration with the highest lateral hamstrings force resulted in the most posterior translation of the medial lowest point, most anterior translation of the lateral lowest point, and the highest tibial external rotation of the five loading configurations. As the medial hamstring force ratio increased, the medial lowest point shifted anteriorly, the lateral lowest point shifted posteriorly, and the tibia rotated more internally. The results of this study, demonstrate that variation in medial-lateral hamstrings force and force ratio influence tibiofemoral transverse kinematics and quadriceps loads required to extend the knee. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1707-1715, 2016.