Background: The consumption of high amounts of fructose is associated with metabolic diseases. However, the underlying mechanisms are largely unknown. Objective: To determine the effects of high fructose intake on plasma metabolomics. Study design: We enrolled 12 healthy volunteers (six lean and six obese women, age 24–35 years) in a crossover intervention study. All participants carried out three diets: (1) low fructose (<10 g/day); (2) high fructose (100 g/day) from natural food sources (fruit); and (3) high fructose (100 g/day) from high fructose syrup (HFS). Outcome measures: The primary outcome was changes in plasma metabolites measured by targeted metabolomics. Results: High compared to low fructose diets caused a marked metabolite class separation, especially because of changes in acylcarnitine and lysophosphatidylcholine levels. Both high fructose diets resulted in a decrease in mean acylcarnitine levels in all subjects, and an increase in mean lysophosphatidylcholine and diacyl-phosphatidylcholine levels in obese individuals. Medium chain acylcarnitines were negatively correlated with serum levels of liver enzymes and with the fatty liver index. Discussion: The metabolic shifts induced by high fructose consumption suggest an inhibition of mitochondrial β-oxidation and an increase in lipid peroxidation. The effects tended to be more pronounced following the HFS than the fruit diet.