The objective of the study was to evaluate the ability of a nonlinear ultrasound technique, the so-called nonlinear resonant ultrasound spectroscopy (NRUS) technique, for detecting early microdamage accumulation in cortical bone induced by four-point bending fatigue. Small parallelepiped beam-shaped human cortical bone specimens were subjected to cyclic four-point bending fatigue in several steps. The specimens were prepared to control damage localization during four-point bending fatigue cycling and to unambiguously identify resonant modes for NRUS measurements. NRUS measurements were achieved to follow the evolution of the nonlinear hysteretic elastic behavior during fatigue-induced damage. After each fatigue step, a small number of specimens was removed from the protocol and set apart to quantitatively assess the microcrack number density and length using synchrotron radiation micro-computed tomography (SR-µCT). The results showed a significant effect of damage steps on the nonlinear hysteretic elastic behavior. No significant change in the overall length of microcracks was observed in damaged regions compared to the load-free control regions. Only an increased number of shortest microcracks, those in the lowest quartile, was noticed. This was suggestive of newly formed microcracks during the early phases of damage accumulation. The variation of nonlinear hysteretic elastic behavior was significantly correlated to the variation of the density of short microcracks. Our results suggest that the nonlinear hysteretic elastic behavior is sensitive to early bone microdamage. Therefore NRUS technique can be used to monitor fatigue microdamage progression in in vitro experiments.Read less <