Whole body vibrations (WBV) are a potential source for low back pain. The mechanism that associates external WBV and internal spinal overload is still unknown, but might be investigated through joint approaches using numerical simulations and in vitro studies. Knowledge about the mechanical behaviour of the spinal structures is essential for this approach. This study aims to determine the influence of frequency and loading magnitude on the spinal stiffness and of loading magnitude on the numbers of cycles to failure. Functional spinal units (L2-L3, L4-L5) from three donor groups were collected: Midlife Male, Midlife Female and Young Male. Characteristic parameters e.g. endplate area (AREA) and bone mineral density (BMD) were determined. Vertebral Capacity (VC), the product of both, was used as principal influencing factor. In vitro testing was performed in tempered saline solution. 6 specimens underwent ultimate strength testing, enabling comparison of the test pool with previously published measurements. 36 specimens endured cyclic testing. Tests started with non-destructive loading (0.005-12 Hz) in axial compression (<2 kN) and shear (<0.3 kN), followed by fatigue loading (<300,000 cycles, 5°Hz), either in shear (n = 6) or axially (n = 30). For axial loading, the specimens were assigned to three groups with different peak-to-peak loads (0-2 kN, 0-3 kN and 1-3 kN). High BMD of the Young Male group and small AREA of the Midlife Female group resulted in greater VC (37%) for the former group (p < 0.001). Ultimate strength results were similar to former studies. Analysis of stiffness parameters revealed that stiffness is non-linear in both load directions, axial stiffness was reduced by shear preload (p < 0.001), anterior shear stiffness was larger with a superimposed anterior offset (p = 0.005). Midlife Females exhibited a 23% smaller axial stiffness than Midlife Males (p < 0.001); shear stiffness for Young Males was larger than for Midlife Males (p = 0.005). Stiffness increased with frequency (axial, 19%, p < 0.001 and shear, 25%, p < 0.001). For the 0-2 kN fatigue loading, endplate failure occurred occasionally (4 of 8), and frequently for 0-3 kN (10 of 13; 1 excluded). Loading with high peak but small amplitude (1-3 kN) lead to occasional specimen failure (4 of 7; 1 excluded). Higher loading amplitudes reduced cycles to failure compared to smaller amplitude, even though the maximum peak (3 kN) was the same. The characteristic creep curves for shear fatigue loading exhibited minor and major discontinuities and separation of the annulus from the endplates, and failure of the bony posterior elements (1 excluded) appeared. The specimens exhibited a large inner- and intra-group variation in VC. AREA serves as a scaling factor for converting axial compressive force to stress. BMD is known to be related to Young’s modulus of the vertebral bony structure. The testing of parameters delivered a broad database for numerical analysis, focusing on passive loading as whole body vibrations and load bearing, rather than different postures and voluntarily performed flexion. A cyclic load magnitude of 40-50% of the respective ultimate strength results in fatigue failure for most axially loaded specimens and all shear loaded specimens. Axial failure appears to be predictable using VC; however no correlation was found for shear failure. Other than peak loading, the amplitudes of the cyclic loading dominantly influence fatigue fracture.