Objective: This study investigated whether diet-induced weight loss alters indices of in vivo postprandial fat uptake in adipose tissue (AT) and whether these changes are associated with weight regain in adults with overweight and obesity. Methods: In this randomized controlled trial, 16 (6 male) individuals (BMI: 28-35 kg/m2) were randomized to either a low-calorie diet (1,250 kcal/d) for 12 weeks or a very-low-calorie diet (500 kcal/d) for 5 weeks (weight loss [WL] period) followed by a 4-week weight-stable (WS) period (together, the dietary intervention [DI] period) and a 9-month follow-up period. Arteriovenous difference measurements combined with stable isotope labeling ([U-13C] palmitate) of a mixed meal were used to determine postprandial fatty acid uptake in AT. Results: Body weight was significantly reduced during the WL period (-8.2±0.6 kg, P<0.001), remained stable during the WS period (0.4±0.3 kg, P=0.150), and increased during follow-up (3.5±0.8 kg, P=0.001). Meal-derived in vivo fatty acid uptake dynamics across AT and expression of genes important for fatty acid uptake, storage, and release were not significantly changed during the DI period. Conclusions: Subcutaneous AT does not appear prone to enhanced meal-derived fatty acid uptake after weight loss, nor were fatty acid uptake dynamics detected as related to weight regain.