Background/Objectives: Sweeteners and sweetness enhancers (S&SEs) have been proposed to impair energy metabolism acutely and over time. Methods: This sub-study investigated the acute effects of a mixture of acesulfame potassium and cyclamate (Ace-K/Cyc) versus water on postprandial substrate oxidation, energy expenditure, and glucose and insulin concentrations at baseline, after a 2-month weight loss period, and after a 4-month weight loss maintenance period including or excluding S&SEs in the diet. In both groups, allowed added sugar intake was <10 energy-%. A total of 26 participants (18–65 years; BMI ≥ 25.0 kg/m2) were recruited from the 1-year randomized controlled SWEET trial, and 16 completed the final test day. Fasting measurements were taken (ventilated hood; blood samples) before a standardized breakfast. Hereafter, postprandial measurements were taken until time point 250 min. At time point 120 min, a test drink containing either Ace-K/Cyc or water was served. Result: There were no differences between the two groups in fasting or postprandial fat oxidation (primary outcome), carbohydrate oxidation, energy expenditure, glucose, or insulin (secondary outcomes) on or across the three test days (p > 0.05). Conclusions: There were no statistically significant differences between Ace-K/Cyc and water in postprandial fat oxidation, carbohydrate oxidation, energy expenditure, or glucose or insulin either acutely, after a weight loss period, or after a 4-month period including or excluding S&SEs in the diet. The low number of participants should, however, be considered a limitation of our conclusions.