Objective To study the influence of the preload on the stability of anterior cruciate ligament reconstruction by hamstring tendon knot embedding method. Methods Eighteen fresh frozen porcine distal femurs were drilled to form bottleneck-like bone tunnels. A knot was tied in the middle part of the hamstring tendons, and the double-stranded tendons were inserted and embedded in the bone tunnel. The tendons were preloaded at 50N?100N and 200N three times respectively before test. Pull-out experiments were performed on bio-material test machine. Results In 50N preload group, the displacement of 400 Newton load was higher than the other two groups and the stiffness was lower than the other two. The maximum load was similar with the 100N group (P>0.05), but higher than 200N group (P<0.01). There was no statistically significant difference between the 100N preload group and 200N preload group with regard to 400N displacement and stiffness (P>0.05). Conclusions The preload has an impact upon peak load?displacement and stiffness in hamstring tendon knot embedding fixation. When the preload was applied between 100 Newton and 200 Newton, the stiffness increased significantly, whereas the maximum load decreased slightly.