OBJECTIVE To investigate the metabolic changes of mice serum after loaded swimming and to provide a basis for the study of anti-fatigue functional food. METHODS The male Kunming mice were randomly divided into four group, fed an AIN-93 diet for 14 days, and forced to swim for 30, 60 or 120 min, respectively, with a load on their tails. The mice were executed after swimming immediately and the changes of serum metabolic profiles were analyzed using metabolomic approach. The spectrum was acquired by using Carr Purcell Meiboom Gill (CPMG) or Longitudinal Eddy Current Delay (LED) sequence, and transformed into 1H NMR spectrogram via Fourier transformation. All the data were analyzed by principal component analysis by using the SIMCA-P+ software. RESULTS The serum metabolic profiles changed significantly after loaded swimming. Serum beta-hydroxybutyric acid, acetate, lactate, lipid were increased and glucose, choline, phosphorylcholine, alanine and phosphatidylcholine decreased. These changes were time dependent. CONCLUSION The changes of serum metabolic profiles after loaded swimming were time dependent, especially for lipid metabolite.Further study based on the interaction of choline and lipid metabolism may contribute to understand the mechanism of fatigue.