Pairs of frozen human patellar tendon-bone (PTB) ligament allografts were exposed to either 0 or 4 Mrad of gamma irradiation sterilization, the latter value based on recent reports suggesting higher dosage levels for adequate sterilization against the human immunodeficiency virus. All specimens were subjected to three levels of loading: lower functional loads, higher functional loads, and failure. Lower functional loads were simulated by performing in vitro static and cyclic creep tests, similar to loads that the surgeon and patient would apply before and after implantation, respectively. Higher functional loads, simulating moderate activities of daily living, were represented by the slope of the linear portion of the force-elongation curve or linear stiffness. Failure or trauma was then simulated by failing the grafts in tension at a high strain rate. We found that the irradiation treatment shortened the tendon by only 0.6 mm, which was nevertheless statistically significant (p < 0.01). By contrast, 4 Mrad did not significantly alter either static or cyclic creep (p > 0.05) at lower functional loads. Instead, irradiation produced the greatest changes during failure testing, reducing both the graft's linear stiffness by 12% (p < 0.025) and maximum force by 26% (p < 0.001). Although our data do not describe how an allograft might perform during the early healing and later collagen-remodelling phases, such in vitro studies remain important if we are to optimize allograft properties before arthroscopic anterior and posterior cruciate ligament reconstruction.