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The equine hoof wall has a complex, hierarchical structure that can inspire designs of impactresistant materials. In this study, we utilized micro-computed tomography (µ-CT) and serial blockface scanning electron microscopy (SBF-SEM) to image the microstructure and nanostructure of the hoof wall. We quantified the morphology of tubular medullary cavities by measuring equivalent diameter, surface area, volume, and sphericity. High-resolution µ-CT revealed that tubules are partially or fully filled with tissue near the exterior surface and become progressively empty towards the inner part of the hoof wall. Thin bridges were detected within the medullary cavity, starting in the middle section of the hoof wall and increasing in density and thickness towards the inner part. Porosity was measured using three-dimensional (3D) µ-CT, twodimensional (2D) µ-CT, and a helium pycnometer, with the highest porosity obtained using the helium pycnometer (8.07%), followed by 3D (3.47%) and 2D (2.98%) µ-CT. SBF-SEM captured the 3D structure of the hoof wall at the nanoscale, showing that the tubule wall is not solid, but has nano-sized pores, which explains the higher porosity obtained using the helium pycnometer. The results of this investigation provide morphological information on the hoof wall for the future development of hoof-inspired materials and offer a novel perspective on how various measurement methods can influence the quantification of porosity.