Mechanical testing of biological tissues often utilizes previously frozen samples but there are conflicting reports about the impact of freezing on mechanical properties. There are no current studies examining the impact of freezing on the interlamellar matrix in the annulus fibrosus of intervertebral disc. We quantified the freezing-induced changes in the interlamellar mechanical properties of degenerated human and non-degenerated bovine annulus fibrosus tissues. Fresh human annulus fibrosus samples were obtained from surgical cases involving disc degeneration (loss of disc height with no deformities), degenerative scoliosis, degenerative spondylolisthesis, and isthmic spondylolisthesis, while bovine samples served as controls. The fresh samples were tested immediately following tissue resection whereas frozen samples were stored at −20 °C for three weeks before testing. An interlamellar peel test was performed where the lamellae were peeled at 0.5 mm/s until complete separation. In paired (same individual) and unpaired (different individuals) degenerated human samples, frozen tissues showed statistically significant decreases:​ Peel Stiffness (50 % and 36 %), Peel Strength (37 % and 45 %), and Standard Deviation of Peel Stress in the Peel Region (57 % and 55 %), with decreasing trends for Peel Toughness (41 % and 42 %), compared to fresh tissues. In contrast, non-degenerated bovine paired samples showed no significant differences between fresh and frozen groups. The results suggest that freezing negatively impacts interlamellar matrix properties in degenerated human annulus fibrosus, which may be exacerbated by existing tissue defects. In the future, annulus fibrosus samples may be best tested fresh to fully capture mechanical differences of the interlamellar matrix between clinical groups.