Bone is unusual among structural materials as it is alive and capable of self-repair. Fatigue-induced microdamage is repaired by bone remodelling, but if damage accumulates too quickly, or remodelling is deficient, fatigue failure may result. Fatigue is thought to contribute to both stress and fragility fractures which are of major clinical importance. Despite this, we do not fully understand the nature of fatigue damage in bone. Human rib sections, containing microcracks stained with basic fuchsin, were serially sectioned and microcracks identified and reconstructed in three dimensions using computer software. Microcracks were elliptical in shape, 400 μm long and 100 μm wide, typical of a transversely isotropic material. Chelating agents which bind Ca²⁺ were found to label microcracks in rib, as well as mineralising bone surfaces and resorption sites, suggesting that microcracks are Ca²⁺ ion-lined discontinuities in the hydroxyapatite matrix. Ca²⁺ ions were exposed by scratching the surface of bovine bone specimens and labelled with chelating agents in sequence. The optimal four agent sequence was:​ alizarin, xylenol orange, calcein and calcein blue. Two dye sequences were used to differentiate between pre-existing and test-induced microdamage in bovine samples fatigue tested in compression and longer sequences labelled microcrack growth. Microcrack dimensions can be used to calculate stress intensity values and, together with fatigue test data, can aid theoretical models to predict fatigue failure in bone.