This study aimed to assess the effects of Anti-fatigue Decoction (AFD) against central fatigue by observing the behaviors and serological indicators of rats modeled by the modified multiple platform method (MMPM) after drug intervention. Grip strength measurements were used to evaluate the muscle strength of rats. The open field test was utilized to assess anxiety-like behavior, while the Morris water maze test was conducted to evaluate the memory function of the rats. Following the behavioral assessments, rat serum samples were collected to measure the concentrations of corticosterone (CORT) and lactic acid (LAC). The concentration of LAC was determined using the colorimetric method, while the concentration of CORT was measured using the enzyme-linked immunosorbent assay (ELISA) method. Compared to the blank control group, following MMPM modeling, rats exhibited significant reductions in grip strength and impaired ability to memory. The serum analysis revealed increased levels of LAC and CORT in the model group rats. AFD can noticeably reverse these adverse changes to a certain extent. These findings highlight the positive effects of AFD and coenzymeQ10 on physical and cognitive abilities and alterations in serum biomarker levels of central fatigue rats.