Appropriate graft tension and secure graft incorporation in bone tunnels are essential for successful anterior cruciate ligament (ACL) reconstruction using hamstring tendon autografts. Permanent viscoplastic elongation in response to cyclic loading in the early postoperative period and the interposition of suture material in the tendon-bone interface might negatively affect graft function and rigid graft incorporation in the bone tunnels. A modified Prusik knot is an alternative option to the commonly used whipstitch technique for soft tissue fixation in ACL reconstruction. This is a controlled laboratory study. Sixteen formalin-fixed human cadaver semitendinosus tendons were armed with a modified Prusik knot or a whipstitch and tested in a load-to-failure test with a constant displacement rate of 1 mm/s, 14 in the cyclic loading test with 100 cycles from 10 to 50 N followed by 100 cycles from 10 to 75 N. The modified Prusik knot showed smaller force-induced displacements and higher stiffness of the entire construct in the load-to-failure test. Smaller preconditioning displacements were the only significant differences in the cyclic loading test. The modified Prusik knot has equal or superior mechanical properties and provides a larger area in the tendon-bone interface without suture material compared with the whipstitch technique.