The antioxidant and antifatigue activities of two peptides of <5 kDa were determined, that is, loach peptide A (LPA, from a papain digestion) and loach peptide B (LPB, from a Flavorzyme digestion). Their degrees of hydrolysis were 21 ± 0.21 and 35 ± 0.32%, respectively. LPA fraction mainly possessed peptides of 1000 < MW < 3000 Da (65.41%), whereas LPB mainly possessed peptides of 500 < MW < 1000 Da (58.27%). LPA fraction contained 116.3 mg amino acid residues/g loach peptide powder of branched-chain amino acids, 1.42-fold that in LPB. LPA had stronger in vitro antioxidant activity than LPB. Compared with LPB, LPA increased swimming time more effectively and reduced blood urea nitrogen (BUN) and liver malonaldehyde (MDA) levels in mice, although both of them had significant antifatigue effects compared to the control (P < 0.05). Pearson correlation analysis showed that the antifatigue activity of loach peptide was highly correlated with its antioxidant activities.