Metallothioneins (MTs) are ubiquitous, multifunctional proteins with key roles in metal homeostasis and redox regulation. Their involvement in cellular energy metabolism is evident from the observation that metallothionein- knockout (MTKO) mice become moderately obese. Transcriptomic studies have also indicated that several genes associated with energy metabolism are differentially expressed in these mice. Although single varying metabolites have been reported, the roles of MTs on a global metabolic level have not been investigated before. In this study, an untargeted, hypothesis-generating metabolomics approach was used to identify and report all metabolites that differ in relative concentration between MT1?2KO, MT3KO and wildtype (WT) mice before and after an exercise (1 h swim) perturbation. PCA and univariate results confirmed that the metabolism of the MTKO mice differs from the WT during unchallenged conditions and hypothetically pointed to increased anabolic activity which could contribute to their previously reported tendency to become obese. Furthermore, the metabolic differences observed in the liver after the 1 h swim indicated that catabolic activity might be impaired in these mice, which could be a consequence of a dysfunction of a common catabolic signal. The MT3KO mice did not show the same metabolic pattern as the MT1?2KO mice as most metabolite concentrations in the brain of these mice were lower after the 1 h swim. The reported metabolic variation contributes to better understand the diverse functionality of these ubiquitous proteins on a global phenotypic level. ; http://link.springer.com/journal/11306