The complex, direction-dependent, poro-viscoelastic properties of the intervertebral disc (disc) suggest that investigations of the six degree of freedom (6DOF) behaviour may be susceptible to inter-test variation in mechanical response if the disc does not return to initial conditions between loading directions. No studies have quantified the effects of sequential multi-directional loading on the consistency of the compressive response of the disc throughout a 6DOF testing protocol. Therefore, the objective of this study was to determine the effect of 6DOF loading on the compressive properties (stiffness and phase angle) of human discs, as evaluated by a reference compression test performed after each single DOF test. Fourteen intact human functional spinal units (FSU) were tested in each of ±6DOFs (shear directions followed by bending and compression) across four orders of magnitude loading frequencies (0.001–1 Hz), followed by reference compression tests while subjected to physiological preload, hydration, and body temperature conditions in a hexapod robot. Repeated measures ANOVA revealed significant within-subjects effects between the reference compression tests for modulus (p<0.001), stiffness (p<0.001), and phase angle (p=0.008). Significant post-hoc pairwise comparisons were initially seen between the control and other reference compression tests for stiffness and modulus after the shear DOFs, however, no significant differences were present after the final reference compression test compared to control. More pronounced effects were seen for stiffness in comparison to modulus and phase angle. These effects may be due to three potentials factors, which include the sequence of testing, the cohort of degenerative specimens, and/or cumulative creep due to the constant application of a follower load. While the sequence of test directions was chosen to minimise the biphasic effect, there may be other sequences, which could result in minimal changes in compressive properties.