Background: Intramyocellular triacylglycerol (IMTG) is utilized as metabolic fuel during exercise and is linked to insulin resistance, but the long-term effect of weight loss strategies on IMTG among participants with abdominal fat, remain unclear. Methods: In an 18-month trial, sedentary participants with abdominal fat/dyslipidemia were randomized to either a low-fat (LF) or Mediterranean/low-carbohydrate (MED/LC) diet (including 28gday-1 of walnuts). After 6-months, the participants were re-randomized to moderate intense physical activity (PA+) or non-physical activity (PA-). Magnetic resonance imaging (MRI) was used to quantify changes of IMTG, abdominal sub-depots, hepatic and intermuscular fats. Results: Across the 277 participants [86% men, age = 48 years, body-mass-index (BMI) = 31kg/m2, visceral fat = 33%] 86% completed the 18-m trial. At baseline, women had higher IMTG than men (3.4% vs. 2.3%, p\u3c0.001) and increased IMTG was associated with aging and higher BMI, visceral and intermuscular fats, HbA1c%, HDL-c and leptin(p\u3c0.05), but not with intra-hepatic fat. After 18 month of intervention and a -3 kg mean weight loss, participants significantly increased IMTG by 25%, with a distinct effect in the MED/LCPA+ group as compared to the other intervention groups (57% vs. 9.5–18.5%, p\u3c0.05). Changes in IMTG were associated with visceral and intermuscular fat, metabolic syndrome, insulin and leptin (p\u3c0.05 for all), however, these associations did not remain after adjustment for visceral fat changes. Conclusions: Lifestyle strategies differentially affect IMTG accumulation; combination of exercise with decreased carbohydrate/increased unsaturated fat proportion intake greatly increase IMTG. Our findings suggest that increased IMTG during diet-induced moderate weight loss may not be directly related to cardiometabolic risk. Trial registration: ClinicalTrials.gov NCT0153072
