Hoof horn, which forms the capsule at the lower part of the legs of many grazing animals including equids (horses, donkeys and mules), is a composite natural material based on α-keratin. Its function is influenced by the tubular and intertubular material and is modulated by the moisture content. There is a requirement to adopt a standard approach to drying regimes and sampling protocols in order to make progress in understanding how the biomechanical properties of hoof horn are related to its structure. In this work the stiffness of donkey hoof has been examined using a three point bending technique and the effect of hydration has been investigated. Also the tubule density properties of this hoof horn material are reported.