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We explored fever induced by baker's yeast in rats using NMR coupled with GC–MS.Potential biomarkers relating to the fever induced by baker's yeast were selected.We examined metabolomic change in aspirin-treated rat by NMR and GC–MS.GABA and glucose were likely involved in the antipyretic pathway of aspirin.We found a useful method to investigate pathological models and to evaluate drugs.A metabolomic investigation of baker's yeast-induced fever in rats was carried out. Plasma derived from Sprague-Dawley rats treated by subcutaneous administration of 20% (w/v) baker's yeast was analyzed using gas chromatography–mass spectrometry (GC–MS) and nuclear magnetic resonance (NMR). Statistical data analysis using t-test and orthogonal partial least-squares discriminant analysis revealed many significant changes in the metabolic data in the plasma of the fever group. Clear separation was achieved between the fever and control groups. Seventeen marked metabolites were found in the fever group. The metabolites, which include amino acids, carbohydrate, organic acids, and fatty acids, mostly contributed to the discrimination of plasma samples from the control and fever groups. These results suggested that fever may involve in the perturbation of amino acid metabolism coupled with energy metabolism, lipid metabolism, and glycometabolism. After determining the antipyretic effects of aspirin on the fever group, four metabolites in the fever rat plasma were found to be signally regulated and recognized as potential biomarkers, including 3-hydroxybutyric acid, gamma-aminobutyric acid, glucose, and linoleic acid. The metabolic relationships that possibly exist between these potential biomarkers were speculated, and the mechanism of baker's yeast-induced fever was illustrated based on the metabolic relationships. This study found that metabolomic approaches such as GC–MS and NMR could be used as potential powerful tools to investigate the biochemical changes and mechanisms in certain pathological states at the metabolism level.