• eNOS−/− mice fed a high fat diet develop NAFLD and accumulate intra‐abdominal and liver fat and display altered lipid composition.• Metformin results in decreased lipid accumulation and partly mitigates the development of NAFLD.• In vivo and ex vivo 1H‐MRS can quantify hepatic steatosis and response to treatment in this model. Here, NOS3 is linked to metabolic dysfunction-associated steatotic liver disease.