Ageing, disease and osteoporosis treatment have been linked to accumulation of microdamage, which is caused by repetitive loading and may eventually causes fatigue failure of bones. Post-hoc investigations for in vivo loading and in vitro experiments have been developed to better understand microdamage formation. In this context, previous studies were not able to discriminate the effects caused by structural changes of the trabecular network from differences of tissue/material properties on microdamage formation. In the present study a fatigue test protocol was established to induce microdamage at a defined tensile stress state of individual trabeculae. Further, a thorough analysis of microdamage analysis was presented for 2D and 3D confocal images, enabling a comparison between the tissue and the meso-scale. Eight individual trabeculae were tested for 1500 cycles, six for 2100 cycles and seven for 3000 cycles (close to failure). Microdamage increased slowly from 1500 to 2100 cycles and showed a rapid increase at 3000 cycles. Diffuse damage was mainly present, although also linear microcracks were visible at 2100 and 3000 cycles. Average microcrack length was 93 µm and diffuse damage density was 4.4% for samples tested for 3000 cycles, comparable to previous studies on trabecular bone cores. Only one to three large microdamage sites were observed in the central region, connected to the trabecular surface with small straight cracks. The presented procedure is a first step to better understand how microdamage formation is influenced by material properties in aged and diseased bone, independently of deteriorated trabecular microarchitecture.