Healthy function of intervertebral discs (IVDs) depends on their tissue mechanical properties. Native cells embedded within IVD tissues are responsible for building, maintaining, and repairing IVD structures in response to genetic, biochemical, and mechanical signals. Organ culturing provides a method for investigating how cells respond to these stimuli in their natural architectural environment. The purpose of this study was to determine how organ culturing affects the mechanical characteristics of functional spine units (FSUs) across the entire range of axial loading, including the neutral zone (NZ), using a rat tail model. Rat tail FSUs were organ cultured at 37 °C in an unloaded state in standard culture media for either 1-day (n = 8) or 6-days (n = 12). Noncultured FSUs (n = 12) were included as fresh control specimens. Axial mechanical properties were tested by applying cyclical compression and tension. A novel mathematical approach was developed to fully characterize the relationship between load, stiffness, and deformation through the entire range of loading. Culturing FSUs for 1-day did not affect any of the axial mechanical outcome measures compared to noncultured IVDs; however, culturing for 6 days increased the size of NZ by 112% and decreased the stiffness in NZ, compressive, and tensile regions by 53%, 19%, and 15%, respectively, compared to noncultured FSUs. These results highlight the importance of considering how the mechanical integrity of IVD tissues may affect the transmission of mechanical signals to cells in unloaded organ culturing experiments.