Interstitial fluid (ISF) surrounds the cells and tissues of the body. Since ISF has molecular components similar to plasma, as well as compounds produced locally in tissues, it may be a valuable source of biomarkers for diagnostics and monitoring. However, there has not been a comprehensive study to determine the metabolite composition of ISF and to compare it to plasma. In this study, the metabolome of suction blister fluid (SBF), which largely consists of ISF, collected from 10 human volunteers was analyzed using untargeted high-resolution metabolomics (HRM). A wide range of metabolites were detected in SBF, including amino acids, lipids, nucleotides, and compounds of exogenous origin. Various systemic and skin-derived metabolite biomarkers were elevated or found uniquely in SBF, and many other metabolites of clinical and physiological significance were well correlated between SBF and plasma. In sum, using untargeted HRM profiling, this study shows that SBF can be a valuable source of information about metabolites relevant to human health.