While the effects of disc degeneration on compression and rotation motions have been studied, there is no data for shear loading. Clinical research has shown that those with low back pain (a potential consequence of degeneration) experience a 75% greater lateral shear force than those without it. Therefore, the aim was to compare the effect of degeneration on spine segment stiffness and phase angle in each of six degree of freedom (6DOF) loading directions. Fourteen intact functional spinal units (FSU) were dissected from human lumbar spines (mean (SD) age 76.2 (11) years, Thompson grades 3 (N = 5, mild), 4 (N = 6, moderate), 5 (N = 3, severe)). Each FSU was tested in ±6DOFs while subjected to a physiological preload, hydration, and temperature (37°C) conditions in a hexapod robot. A one-way ANOVA between degenerated groups was performed on stiffness and phase angle for each DOF. Significant differences in stiffness were found between mild and moderate degenerative groups in lateral shear (p = 0.001), and axial rotation (p = 0.001), where moderate degeneration had decreased stiffness. For phase angle, significant differences were seen in anterior shear (p = 0.017), and axial rotation (p = 0.026), where phase angle for mild degeneration was less than moderate. Trends of stiffness and phase angle changes between degenerative groups were similar within each DOF. Clinically, the identification of the DOFs that are most affected by degeneration could be used in rehabilitation to improve supplemental stabilization of core muscle groups.