Proton nuclear magnetic resonance (¹H-NMR) spectroscopy of plasma provides a global metabolic profiling method which shows promise for clinical diagnostics. However, cross-sectional studies are complicated by a lack of understanding of intra-individual variation, and this limits experimental design and interpretation of data. The present study determined the diurnal variation detected by ¹H NMR spectroscopy of human plasma. Data reduction methods revealed 3 time-of-day metabolic patterns, which were associated with morning, afternoon and night. Major discriminatory regions for these time-of-day patterns included the various kinds of lipid signals (-CH₂- and -CH₂OCOR) and the region between 3-4 ppm heavily overlapped with amino acids which had -CH and -CH₂. The phasing and duration of time-of-day patterns were variable among individuals, apparently due to individual difference in food processing/digestion and absorption and clearance of macronutrient energy sources (fat, protein, carbohydrate). The times of day which were most consistent among individuals, and therefore most useful for cross-sectional studies, were fasting morning (8:30-9:30), post-prandial afternoon (14:30-16:30) and nighttime samples (4:30-5:30). Importantly, the integrated picture of metabolism provided by ¹H-NMR spectroscopy of plasma suggests that this approach is suitable to study complex regulatory processes including eating patterns/eating disorders, upper gastrointestinal functions (gastric emptying, pancreatic, biliary functions) and absorption/clearance of macronutrients. Hence, ¹H-NMR spectroscopy of plasma could provide a global metabolic tolerance test to assess complex processes involved in disease, including eating disorders and the range of physiologic processes causing dysregulation of energy homeostasis.