Bone tissue is subjected to increased mechanical stress during high-intensity work. Inadequate bone remodeling reparability can result in the continuous accumulation of microdamage, leading to stress fractures. The aim of this work was to investigate the characteristics and repair mechanisms of tibial microdamage under several degrees of overload. Also, we aimed at better understanding the effects of overload on the multi-scale structure and mechanical properties of bone. Sixty 5-month female rats were divided into three groups with different time points. Micro-CT was used to evaluate the three-dimensional microstructure, and three-point bending, quasi-static fracture toughness and creep mechanical test were carried out to evaluate the mechanical properties. SEM was used to observe the morphological characteristics of fracture surfaces. Section staining was used to count the microdamage parameters and numbers of osteoblasts and osteoclasts. The microarchitectures of cancellous and cortical bones in the three overload groups showed different degrees of damage. Overload led to a messy crystal structure of cortical bone, with slender microcracks mixed in, and a large number of broken fibers of cancellous bone. The properties associated with the elastic plasticity, fracture toughness, and viscoelasticity of cortical bone reduced in three groups, with that corresponding to day 30 presenting the highest damage. The accumulation of microdamage mainly occurred in the first 14days, that is, the crack density peaked on day 14. Peak-targeted bone remodeling of cortical and cancellous bones occurred mainly between days 14 and 30. The influence of overload mechanical environment on bone quality at different time points was deeply investigated, which is of great significance for the etiology and treatment of stress fractures.