Background Initial graft tension influences clinical results of anterior cruciate ligament reconstruction. Hypothesis Under repetitive loading conditions, the effect of initial graft tension on the biomechanical behavior of the femur-graft-tibia complex may depend on the graft and the fixation. Study Design Ex vivo biomechanical laboratory study. Methods After anterior cruciate ligament reconstruction, initial graft tension of 20, 80, or 140 N was applied to the complex for 2 minutes. Then, a cyclic force-relaxation test was performed for 5000 cycles so that the graft was stretched by 2 mm. Results In a patellar tendon graft with interference screws, the average peak load values at the 5000th cycle were 105, 157, and 205 N for the complexes with initial tension of 20, 80, and 140 N, respectively. In a flexor tendon graft with interference screws, the values were 27, 41, and 39 N. In a flexor tendon graft with Endobutton fixation, the values were 17, 40, and 77 N. Conclusions Considering the tension of the normal anterior cruciate ligament (16 to 87 N), an initial tension of 20 N appears to be high enough for a patellar tendon graft. For a flexor tendon graft with interference screws, an increase in initial tension above 80 N has no biomechanical advantages.