The investigations on the response of bone tissue under different loading conditions are important from clinical and engineering points of view. In this paper, the influence of nesfatin-1 administration on rat humerus mechanical properties was analyzed. The classical three-point bending and impact tests were carried out for three rat bone groups: control (SHO), the humerus of animals under the conditions of established osteopenia (OVX), and bones of rats receiving nesfatin-1 after ovariectomy (NES). The experiments proved that the bone strength parameters measured under various mechanical loading conditions increased after the nesfatin-1 administration. The OVX bones were most susceptible to deformation and had the smallest fracture toughness. The SEM images of humerus fracture surface in this group showed that ovariectomized rats had a much looser bone structure compared to the SHO and NES females. Loosening of the bone structure was also confirmed by the densitometric and qualitative EDS analysis, showing a decrease in the OVX bones’ mineral content. The samples of the NES group were characterized by the largest values of maximum force obtained under both quasi-static and impact conditions. The energies absorbed during the impact and the critical energy for fracture (from the three-point bending test) were similar for the SHO and NES groups. Statistically significant differences were observed between the mean Fi max values of all analyzed sample groups. The obtained results suggest that the impact test was more sensitive than the classical quasi-static three-point bending one. Hence, Fi max could be used as a parameter to predict bone fracture toughness.