Numerous in vitro studies have examined the initiation and propagation of fatigue injury pathways in the annulus fibrosus (AF) using isolated motion segments; however, the cycle-varying changes to the AF under cyclic biaxial tensile loading conditions have yet to be examined. Therefore, the primary objective of this study was to characterize the cycle-varying changes in peak tensile stress in multilayer AF tissue samples within a range of physiologically relevant loading conditions at subacute magnitudes of tissue stretch up to 100 loading cycles. A secondary aim was to examine whether the stress-relaxation response would be different across loading axes (axial and circumferential) and whether this response would vary across regions of the intervertebral disk (IVD) (anterior and posterior–lateral). The results from the study demonstrate that several significant interactions emerged between independent factors that were examined in the study. Specifically, a three-way interaction between the radial location, magnitude of peak tissue stretch, and cycle rate (p = 0.0053) emerged. Significant two-way interactions between the magnitude of tissue stretch and cycle number (p < 0.0001) and the magnitude of tissue stretch and loading axis (p < 0.0001) were also observed. These findings are discussed in the context of known mechanisms for structural damage, which have been linked to fatigue loading in the IVD (e.g., cleft formation, radial tearing, increased neutral zone, disk bulging, and loss of intradiscal pressure).