Background: In osteoporotic bone, the quality of the bone-to-implant interface is decreased, which may lead to early implant failure. Screw anchorage can be improved by augmentation. This effect is mainly investigated with a pull-out test. To our knowledge, the effect of cement augmentation in an in vivo physiological setup focusing on screw movement has not been investigated to date. The aim of this work was to investigate and compare augmented and native screw behavior in a physiologically related setup. Methods: Twelve fresh-frozen human lumbar vertebrae were divided into two groups. Each vertebra was bilaterally instrumented with either non-augmented or augmented pedicle screw systems and loaded in a recently developed test setup that provided cyclic conditions comparable to a physiological gait. The cyclic loading should test the primary implant stability, comparable to the postoperative period of two months in a worst-case scenario in the absence of osseous remodeling. Screws were tracked optically, and screw movement and failure patterns were observed. Results: Mutual influence between the left and right sides resulted in a successive, rather than simultaneous, failure. Augmentation of the screws in vertebrae with poor bone quality reduced screw subsidence and thus improved the rigidity of the screw-to-implant interface by up to six-fold. The non-augmented condition was significantly related to early screw failure. Conclusions: Pedicle screw system failure involves a complex bilateral-coupled mechanism. The cyclic loading based on physiological conditions during walking has allowed the postoperative conditions and clinical failure mechanisms to be simulated in vitro and clarified. Future implant systems should be investigated with a physiologically related setup.