This study sought to evaluate the effects of prolonged cyclic loading on the tissue-level mechanical properties of the spine, specifically the annulus fibrosus (AF). Methods: Functional spinal units (FSUs) were obtained from porcine cervical spines at the C3-C4 and C5-C6 levels. Following a 15-minute preload of 300N of axial compression, the FSUs were split into 3 groups: the cyclic loading group cycled between 0.35MPa and 0.95MPa for two hours (n=8); the static loading group was compressed at 0.65MPa for two hours (n=10); and a control group which only underwent the 300N preload (n=11). Following loading, samples of the AF were excised to perform intralamellar tensile testing and interlamellar 180° peel tests. Variables analyzed from the intralamellar test were stress and strain at the end of the toe region, stress and strain at initial failure (yield point), Young's modulus, ultimate stress, and strain at ultimate stress. Variables evaluated from the interlamellar tests were lamellar adhesion strength, adhesion strength variability, and stiffness. Results: The analysis showed no significant differences between conditions, however, there was a trend (p=0.059) that cyclically loaded tissues had increased adhesion strength variability compared to the static and control conditions. Discussion: The main finding of this study is that long-duration axial loading did not impact the intra- or interlamellar mechanical properties of the porcine AF. A trend of increased adhesion strength variability in cyclically loaded samples could indicate a potential predisposition to delamination.