Whilst in vitro testing can contribute to a better understanding of the biomechanical interactions at the knee joint, the application of physiological-like muscle forces in vitro remains challenging. One main difficulty seems to be the adequate fixation of the muscle-tendon complex to the mechanical apparatus that provides the forces in vitro. The goal of this study was to compare the ability of different muscle-tendon fixation mechanisms, including a new technique developed to optimise the interface grip of the soft tissues, to reliably transmit physiological in vivo loads through the muscle-tendon complex to the attached bone. The fixations of three quadriceps components in 16 right knees of skeletally mature female merino sheep were loaded to failure using four different fixation techniques (aluminium clamp, freeze clamp, suture technique and a new extension hull technique). Each technique was tested 12 times: 4 times on each individual quadriceps component. A factorial analysis for repeated measurements was undertaken to examine differences between the different fixation techniques. The extension hull technique and the aluminium clamp performed similarly, exceeding the computationally determined physiological forces in all but one trial and achieved higher failure loads than the suture technique. Although the freeze clamp reached the highest mean load to failure, it also failed more often than the extension hull technique. This comparison of the fixation techniques suggests that the new extension hull technique is a suitable fixation method for applying physiological-like muscle loading in an in vitro set-up. It cannot only be handled in a very simple manner, but also possesses a compact, lightweight construction, providing the possibility for the application of more complex loading conditions that include, e.g. the action of multiple muscles of the knee flexor and extensor group concurrently.