While a number of in vitro studies have shown that the tension on an anterior cruciate ligament (ACL) replacement graft at the time of fixation has an affect on joint stability, most in vivo studies have reported little or no long-term difference in outcome. The objectives of this study were to (1) establish a large animal model in which differences in knee stability are present at time-zero after ACL reconstruction with grafts fixed at a low (5 N) and high (35 N) initial tension and to (2) quantitatively determine if these initial effects remain after six weeks of healing and if the tensile properties of an ACL replacement graft are influenced by initial graft tension. Seventeen skeletally mature female Saanan breed goats were used. Using the robotic/UFS testing system, the knee kinematics and in situ forces in the replacement graft in response to an externally applied 67 N anterior-posterior (A-P) tibial load were evaluated at time-zero and after six weeks of healing. Afterward, the femur-ACL graft-tibia complexes (FGTCs) from the six-week group were tested under uniaxial tension so that the stress relaxation and structural properties of the FGTC were obtained. At time-zero, knees fixed with a high initial graft tension could better reproduce the A-P translation of the intact knee in response to the 67 N A-P tibial load. Further, in situ forces in these grafts were also closer to those in the intact ACL under the same external loading condition. After six weeks of healing, the A-P translation of the knee and in situ forces in the replacement grafts became similar for the low and high tension groups, while both were significantly different from controls. Further, the percentage of stress relaxation as well as the stiffness, ultimate load at failure, ultimate elongation at failure, and energy absorbed of the FGTCs for both reconstruction groups were not significantly different from each other, but were significantly different from controls. These results demonstrate that while the high initial graft tension could better replicate the normal knee kinematics at time-zero, these effects may diminish during the early graft healing process.