Physical exercise has potent therapeutic and preventive effects against metabolic disorders. A number of studies have suggested that 5'AMP-activated protein kinase (AMPK) plays a pivotal role in regulating carbohydrate and lipid metabolism in contracting skeletal muscles, while several genetically manipulated animal models revealed significance of AMPK-independent pathways. In order to elucidate significance of AMPK and AMPK-independent signals in contracting skeletal muscles, we conducted a metabolomic analysis which compared the metabolic effects of 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleoside (AICAR) stimulation with the electrical contraction ex vivo in isolated rat epitrochlearis muscles, in which both α1 and α2 isoforms of AMPK and glucose uptake were equally activated. The metabolomic analysis using capillary electrophoresis time-of-flight mass spectrometry detected 184 peaks and successfully annotated 132 small molecules. AICAR stimulation exhibited high similarity to the electrical contraction in overall metabolites. Principal component analysis (PCA) demonstrated that the major principal component characterized common effects whereas the minor principal component distinguished the difference. PCA and a factor analysis suggested a substantial change in redox status as a result of AMPK activation. We also found decrease in reduced glutathione levels in both AICAR-stimulated and contracting muscles. The muscle contraction-evoked influences related to the metabolism of amino acids, in particular: aspartate, alanine or lysine, are supposed to be independent of AMPK activation. Our results substantiate significance of AMPK activation in contracting skeletal muscles and provide novel evidence that AICAR stimulation closely mimics the metabolomic changes in the contracting skeletal muscles.