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Objective: Examine lipid metabolism in liver of rats receiving different caloric and non-caloric sweeteners using magnetic resonance spectroscopy (MRS). Methods: 24 male Wistar rats (343 ± 2 g) were divided into 4 diet groups receiving water (CON), 13% glucose solution (GLU), 13% fructose solution (FRUC), or 0.4% aspartame solution (ASP). After 7 weeks of diet, 1H-[13C] experiments were performed at baseline and after oral administration of 1.5 g [U-13C] Algal lipid mixture per kg body weight. Results: After 7 weeks, body weight of GLU animals was higher compared with ASP animals. Although animals in the GLU and FRUC groups consumed less food compared to CON and ASP animals, their total energy consumption was higher due to the excess intake of calories from sugar in their drinking water. Total liver lipid content was higher in GLU and FRUC animals compared with ASP animals. Four hours after the administration of 13C-labeled lipids, 13C enrichment of intracellular lipids was equally increased in all 4 groups, showing that dietary lipid uptake was not different between groups. Conclusion: The non-caloric sweetener aspartame did not have any effect on liver lipid content or metabolism. Glucose and fructose consumption resulted in increased lipid content in the liver, which was not accompanied by an increased uptake of dietary lipids and is thus likely caused by elevated lipogenesis.