Modifiable lifestyle factors, including exercise and activity energy expenditure (AEE), may attenuate the unfavorable health effects of obesity, such as risk factors of metabolic syndrome (MetS). However, the underlying mechanisms are not clear. In this study we sought to investigate whether the metabolite profiles of MetS and adiposity assessed by body mass index (BMI) and central obesity are inversely correlated with AEE and physical activity. We studied 35 men and 47 women, aged 30-60 years, using doubly labeled water to derive AEE and the Sedentary Time and Activity Reporting Questionnaire (STAR-Q) to determine the time spent in moderate and vigorous physical activity. Proton nuclear magnetic resonance spectroscopy was used for serum metabolomics analysis. Serine and glycine were found in lower concentrations in participants with more MetS risk factors and greater adiposity. However, serine and glycine concentrations were higher with increasing activity measures. Metabolic pathway analysis and recent literature suggests that the lower serine and glycine concentrations in the overweight/obese state could be a consequence of serine entering de novo sphingolipid synthesis. Taken together, higher levels of AEE and physical activity may play a crucial part in improving metabolic health in men and women with and without MetS risk factors.