Objective To predict the tissue-level failure strain of the cortical bone and discuss the effects of different running speeds on the mechanical properties of rat femoral cortical bone. Methods The threshold for cortical bone tissue-level failure strain was assigned, and fracture simulation under three-point bending was performed on a rat femoral finite element model. The predicted load-displacement curves in each simulation were compared and fitted with the experimental data to back-calculate the tissue-level failure strain. Results The cortical bone tissue-level failure strains at different running speeds were statistically different, which indicated that different running speeds had certain impacts on the micromechanical properties of the cortical bone structures. At a running speed of 12 m/min, the cortical bone structure expressed the greatest tissue-level failure strain, and at a running speed of 20 m/min, the cortical bone structure expressed the lowest tissue-level failure strain. Conclusions Based on the changing trends of tissue-level failure strain and in combination with the changes in macro-level failure load and tissue-level elastic modulus of cortical bone structures, the effects of different running speeds on the mechanical properties of cortical bone structures were discussed in this study. The appropriate running speed for improving the mechanical properties of the cortical bone was explored, thereby providing a theoretical basis for improving bone strength through running exercises.