The oral glucose tolerance test (oGTT) is a common tool to provoke a metabolic challenge for scientific purposes, as well as for diagnostic reasons, to monitor the kinetics of glucose and insulin. Here, we aimed to follow the variety of physiologic changes of the whole metabolic pattern in plasma during an oGTT in healthy subjects in a non-targeted reversed-phase UPLC-qTOF mass spectrometric metabolomics approach. We detected 11,500 metabolite ion masses per individual. Applying multivariate data analysis, four major groups of metabolites have been detected as the most discriminating oGTT biomarkers: free fatty acids (FFA), acylcarnitines, bile acids and lysophosphatidylcholines. We found in detail i) a strong decrease of all saturated and monounsaturated FFA studied during the oGTT ii) a significant faster decline of palmitoleate (C16:1) and oleate (C18:1) FFA levels than their saturated counterparts; iii) a strong relative increase of PUFAs in the fatty acid pattern at 120 min; iv) a clear decrease in plasma C10:0, C12:0 and C14:1 acylcarnitine levels. These data reflects the switch from -oxidation to glycolysis and fat storage during the oGTT. Moreover, the bile acids glycocholic acid, glycochenodeoxycholic acid, and glycodeoxycholic acid were highly discriminative showing a biphasic kinetic with a maximum of a 4.5- to 6-fold increase at 30 min after glucose ingestion, a significant decrease over the next 60 min followed by an increase until the end of the oGTT. Lysophosphatidylcholines were also significantly increased. The findings of our metabolomics study reveal detailed insights in the complex physiological regulation of the metabolism during an oral glucose tolerance test offering novel perspectives of this widely used procedure.