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 28g·day-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<0.001) and increased IMTG was associated with aging and higher BMI, visceral and intermuscular fats, HbA1c%, HDL-c and leptin(p<0.05), 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<0.05). Changes in IMTG were associated with visceral and intermuscular fat, metabolic syndrome, insulin and leptin (p<0.05 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 NCT01530724.