Objective To study the physical fatigue behavioral manifestation of mice after sleep interruption (SI)at different timepoints, to provide a stable and reliable animal model for the research and development of general health products. Methods Mice were placed into a rotating drum sleep interferometer for different periods of time (5, 10, and 15 days). Then, the rotating wheel and loaded swimming tests were used to assess the behavioral activities of mice after SI.The hepatic glycogen and serum urea nitrogen levels were used to assess the biochemical index after SI. Results In the rotating wheel exhausted test, the exhausted time of SI in the 5 d, 10 d and 15 d groups were decreased compared with the control group, but with a non-significant difference ( P > 0. 05). In the loaded swimming test, compared with the control group, the mice after SI for 10 and 15 days showed significantly decreased exhaustive time ( P < 0. 05, P < 0. 01), sink frequency and total sink time ( P < 0. 01, P < 0. 01). In addition, our study demonstrated that the content of hepatic glycogen was significantly reduced in mice after SI for 5, 10, and 15 days ( P < 0. 05) compared with the control group,and that the serum urea nitrogen level of mice after SI for 10 and 15 days was increased ( P < 0. 05). Conclusions A mouse model of physical fatigue behavior can be established by sleep interruption using a sleep interruption apparatus set at rotation speed, 20 s per revolution; and rotation frequency, rotating 1 min after 1 min pause for 10 days and 15 days.