Concerns have been raised regarding the influence of hamstring tendon harvest on post-operative knee flexor strength after anterior cruciate ligament (ACL) reconstruction. There is some evidence that harvesting both the semitendinosus and gracilis tendons may lead to greater strength deficits in deep knee flexion when compared to harvesting the semitendinosus alone. PURPOSE: To evaluate the influence of single compared to multiple hamstring tendon harvest on knee flexor strength for patients who had returned to sport following ACL reconstruction surgery. METHODS: Fifty participants were included in the study. They had all returned to regular sporting activity a mean 32.5 months after ACL reconstruction with a four-strand hamstring tendon graft. Patients had either semitendinosus and gracilis (ST/G; n = 30) harvested or semitendinosus alone (ST; n = 20). Isokinetic and isometric hamstring strength were measured, and the standing knee flexion angle used to evaluate functional hamstring strength recovery. RESULTS: No significant differences between the groups were found for any of the strength measures or the standing knee flexion angle. A consistent deficit of between 8 and 22% was observed in the strength of the operated limb when compared to the non-operated, regardless of which tendons were harvested. No relationship was found between the standing knee flexion angle and the isometric hamstring strength results obtained at 105° knee flexion (r2 = 0.034). CONCLUSIONS: The choice between one or two hamstring tendons is unlikely to significantly influence post-operative hamstring strength outcomes in people returning to sport, as both graft options were shown to have similar strength outcomes at two years post-operatively. Persistent hamstring strength deficits in the operated limb compared to the non-operated limb were found which may have implications for post-operative athletic performance. However the functional significance of these deficits is at present unclear. The results also demonstrate the standing knee flexion angle should not be used as a surrogate clinical measure of hamstring strength as it correlated poorly with dynamometric measures of hamstring strength.