The human circulatory system consists of arterial blood that delivers nutrients to tissues, and venous blood that removes the metabolic by-products. Although it is well established that arterial blood generally has higher concentrations of glucose and oxygen relative to venous blood, a comprehensive biochemical characterization of arteriovenous differences has not yet been reported. Here we apply cutting-edge, mass spectrometry-based metabolomic technologies to provide a global characterization of metabolites that vary in concentration between the arterial and venous blood of human patients. Global profiling of paired arterial and venous plasma from 20 healthy individuals, followed up by targeted analysis made it possible to measure subtle (<2 fold), yet highly statistically significant and physiologically important differences in water soluble human plasma metabolome. While we detected changes in lactic acid, alanine, glutamine, and glutamate as expected from skeletal muscle activity, a number of unanticipated metabolites were also determined to be significantly altered including Krebs cycle intermediates, amino acids that have not been previously implicated in transport, and a few oxidized fatty acids. This study provides the most comprehensive assessment of metabolic changes in the blood during circulation to date and suggests that such profiling approach may offer new insights into organ homeostasis and organ specific pathology.