The decellularisation of xenogenic and allogeneic biological grafts offers a promising solution to replacement of the anterior cruciate ligament (ACL). The purpose of this investigation was to determine the biomechanical effects of additional fat reduction and bioburden reduction steps in the decellularisation of porcine super flexor tendon (pSFT). Study 1 investigated the use of acetone or chloroform–methanol as a fat reduction agent. The most effective of these was then carried forward into Study 2, which investigated the use of antibiotics or peracetic acid (PAA) as a bioburden reduction agent. Stress relaxation data was analysed using a Maxwell–Wiechert viscoelastic model and, in addition to classical material properties, the tangent modulus of the toe-region was determined from strength testing data. In both studies, the majority of decellularised groups demonstrated no statistical differences for material properties such as tensile strength and Young’s modulus compared to native controls. Different trends were observed for many of the viscoelastic parameters, but also for the tangent modulus in the toe-region indicating a change in performance at low strains. The most severe deviations from the profile of the native tangent modulus were found to occur in Study 2 when PAA was used for bioburden reduction. Classic material properties (E, UTS etc.) are often used to compare the characteristics of native and decellularised tissues, however they may not highlight changes occurring in the tissues at low strains. In this study, this represented the physiological strains encountered by substitute acellular ACL grafts. Acetone was chosen as the fat reduction step whereas, antibiotics was preferable over PAA as a bioburden reduction step.