Bone is subjected to cyclic loading in vivo through daily physical activities, leading to the accumulation of fatigue microdamage and, possibly, fractures. Therefore, the ability to characterize microdamage accumulation is important for understanding and assessing fracture risk. The overall objective of this study was to evaluate methods for measuring microdamage accumulation in cortical bone using both mechanical and novel, non-destructive histological techniques. During fatigue testing, linear elastic beam theory (LEBT), the secant modulus, the tangent modulus, the unloading modulus, and creep have been used to measure the accumulation of fatigue microdamage via changes in mechanical properties. Mechanical measures for the accumulation of damage in bovine cortical bone during four-point bending fatigue were shown to be highly dependent on the initial specimen modulus and mechanical measure employed. The LEBT modulus was shown to include the combined effect of both elastic (recovered) and creep (accumulated) strain indicating that both the secant modulus and creep should be measured throughout a test to most accurately indicate damage accumulation and account for different damage mechanisms. Moreover, indentation of roller supports resulted in inflated measures of the LEBT modulus degradation and creep. The results of this study suggest that investigations of fatigue microdamage in cortical bone should avoid the use of four-point bending unless no other option is possible. Conventional techniques used to image damage accumulation in cortical bone are inherently invasive, destructive, two-dimensional, and tedious. Therefore, micro-computed tomography was investigated as a possible non-destructive technique using a precipitated barium sulfate contrast agent to label damage. The ratio of the segmented stain volume to bone volume increased from the unloaded control group to specimens loaded in cyclic uniaxial tension to a 5 and 10% reduction in secant modulus. Segmented images showed distinct regions of bright voxels, verified to be due to the presence of BaSO4, which were indicative of fatigue cracks, as well as the accumulation of linear microcracks and diffuse damage. Local variations in mineralization were also measured by micro-CT and suggested to be useful in predicting the susceptibility of tissue to the initiation and accumulation of microdamage.