The purpose of this study was to investigate anti-oxidant effects of loach muscle-derived peptides in vitro and in vivo. Loach muscle peptides were prepared by 4 different methods: boiling (B), enzymatic hydrolysis (E), boiling and enzymatic hydrolysis (BE), alkaline and enzymatic hydrolysis (AE). Two different in vitro analyses, DPPH radical scavenging activity and xanthine-xanthine oxidase-induced superoxide radical scavenging activity, were performed. All the four preparations showed concentration-dependent DPPH radical scavenging activity. However, superoxide radical scavenging activity was found only with AE and E preparations. To evaluate in vivo effects, mice were fed with 10% AE-containing diets for 4 weeks before hepatotoxicity induction with CCl4. In serum glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT) levels, total antioxidant levels, and relative hepatic weight ratio, no evidence for anti-oxidant effects was found with AE indicating the absence of anti-oxidant effect in the in vivo mouse experiment. It needs to be clarified why anti-oxidant activity of loach protein hydrolysates was not evident in vivo. Furthermore, these results suggest that in vivo evaluation is crucial in demonstrating anti-oxidant activities.