While microstructural observations have improved our understanding of possible pathways of herniation progression, no studies have measured the mechanical failure properties of the inter-lamellar matrix (ILM), nor of the adjacent lamellae during progression to herniation. The aim of this study was to employ multiscale, biomechanical and microstructural techniques to evaluate the effects of progressive induced herniation on the ILM and lamellae in control, pre-herniated and herniated discs (N = 7), using 2 year-old ovine spines. Pre-herniated and herniated (experimental) groups were subjected to macroscopic compression while held in flexion (13°), before micro-mechanical testing. Micro-tensile testing of the ILM and the lamella from anterior and posterolateral regions was performed in radial and circumferential directions to measure failure stress, modulus, and toughness in all three groups. The failure stress of the ILM was significantly lower for both experimental groups compared to control in each of radial and circumferential loading directions in the posterolateral region (p < 0.032). Within each experimental group in both loading directions, the ILM failure stress was significantly lower by 36% (pre-herniation), and 59% (herniation), compared to the lamella (p < 0.029). In pre-herniated compared to control discs, microstructural imaging revealed significant tissue stretching and change in orientation (p < 0.003), resulting in a loss of distinction between respective lamellae and ILM boundaries.