Abstract: The behavioral and biochemical indexes were used to study the anti-fatigue effect of Auxis tapeinosom dark muscle protein hydrolysates (DMPH) and were compared with that of dark muscle hydrolysates (DMH) to preliminarily speculated the main anti-fatigue active substance in DMH. The result showed that the content of crude protein in DMPH was significantly higher than DMH (p<0.05) and the total acid concentration was higher than DMH. DMH and DMPH could significantly increase the swimming time of mice and reduce consumption of liver glycogen (p<0.05). They could significantly reduce the content of plasma lactic acid and urea nitrogen in mice and improve the activity of antioxidant enzymes in mice (p<0.05). Both of them had anti-fatigue effect and had no significant differences (p>0.05), and the content of crude protein and total amino acid concentration in DMPH were higher than those of DMH. It could be speculated that amino acids and peptide hydrolysates from dark muscle protein would be the main anti-fatigue active substances in DMH.