BACKGROUND: Protein supplementation has been shown to reduce the increases in intrahepatic triglyceride (IHTG) content induced by acute hypercaloric high-fat and high-fructose diets in humans. OBJECTIVE: To assess the effect of a 12-wk iso-energetic high protein-low carbohydrate (HPLC) diet compared with an iso-energetic high carbohydrate-low protein (HCLP) diet on IHTG content in healthy non-obese subjects, at a constant body weight. DESIGN: Seven men and nine women [mean +/- SD age: 24+/-5 y; BMI: 22.9+/-2.1 kg/m2] were randomly allocated to a HPLC [30/35/35% of energy (En%) from protein/carbohydrate/fat] or a HCLP (5/60/35 En%) diet by stratification on sex, age and BMI. Dietary guidelines were prescribed based on individual daily energy requirements. IHTG content was measured by 1H-magnetic resonance spectroscopy before and after the dietary intervention. RESULTS: IHTG content changed in different directions with the HPLC (CH2H2O: 0.23+/-0.17 to 0.20+/-0.10; IHTG%: 0.25+/-0.20% to 0.22+/-0.11%) compared with the HCLP diet (CH2H2O: 0.34+/-0.20 vs. 0.38+/-0.21; IHTG%: 0.38+/-0.22% vs. 0.43+/-0.24%), which resulted in a lower IHTG content in the HPLC compared with the HCLP diet group after 12 weeks, which almost reached statistical significance (P = 0.055). CONCLUSIONS: A HPLC vs. a HCLP diet has the potential to preserve vs. enlarge IHTG content in healthy non-obese subjects at a constant body weight. TRIAL REGISTRATION: Clinicaltrials.gov NCT01551238.