The serum metabolite profile reflects a great variety of factors including age, gender, diet, exercise, gut microbial metabolism and the presence of disease. Importantly, changes in the serum metabolome may appear prior to the clinical manifestation of disease, provide insight into underlying biological mechanisms and be predictive of disease progression and/or amelioration. Using an animal model and human participants, the serum metabolome of obesity was studied in relations to diet and physical activity. In brief, obese rats consuming coffee had a favorable body composition, lower liver triglycerides and decreased serum concentrations of branched-chain amino acids, which are thought to cause diabetes when present at higher concentrations, compared to controls. In contrast, aspartame consuming rats showed impairments in glucoregulation. Our findings suggested that this might have been a result of aspartame causing an increase in the proportion of gut bacteria that produce propionate, a metabolite known to stimulate hepatic gluconeogenesis. In human subjects, obesity and metabolic syndrome risk factors were associated with lower concentrations of the sphingolipid precursors serine and glycine. Higher activity energy expenditure and physical activity levels showed the opposite association. Physical activity may thus improve on insulin sensitivity by reducing de novo synthesis of sphingolipids and their subsequent accumulation in insulin-sensitive tissues. Exercise also associated with improvements in body weight, lean mass, physical performance and symptom severity, following cancer treatment in head and neck cancer patients. However, none of these factors correlated with their 2-year survival. Instead, the baseline serum metabolite profile differentiated between survivors and nonsurvivors, despite matching for patient characteristics. Thus, serum metabolites show potential as prognostic biomarkers for head and neck cancer patients. Lastly, we found that combining three metabolomics approaches resulted in the most comprehensive coverage of metabolite classes and the most complete description of the phenotype, for women with ovarian cancer. This Chapter also highlighted the need to address the influence of common risk factors on the serum metabolome. Taken together, the work presented in this thesis has provided further insight into the serum metabolite profile of metabolic disease and cancer in the context of diet and physical activity.