ABSTRACT: BACKGROUND:Non-nutritive sweeteners (NNS), especially in form of diet soda, have been linked to metabolic derangements (e.g. obesity and diabetes) in epidemiologic studies. We aimed to test acute metabolic effects of NNS in isolation (water or seltzer) and in diet sodas. METHODS:We conducted a four-period, cross-over study at the National Institutes of Health Clinical Center (Bethesda, Maryland). Thirty healthy adults consumed 355 mL water with 0 mg, 68 mg, 170 mg, and 250 mg sucralose, and 31 individuals consumed 355 mL caffeine-free Diet Rite Cola™, Diet Mountain Dew™ (18 mg sucralose, 18 mg acesulfame-potassium, 57 mg aspartame), and seltzer water with NNS (68 mg sucralose and 41 mg acesulfame-potassium, equivalent to Diet Rite Cola™) in randomized order, prior to oral glucose tolerance tests. Blood samples were collected serially for 130 min. Measures included GLP-1, GIP, glucose, insulin, C-peptide, glucose absorption, gastric emptying, and subjective hunger and satiety ratings. RESULTS:Diet sodas augmented active GLP-1 (Diet Rite Cola™ vs. seltzer water, AUC, p?=?0.039; Diet Mountain Dew™ vs. seltzer water, AUC, p?=?0.07), but gastric emptying and satiety were unaffected. Insulin concentrations were nominally higher following all NNS conditions without altering glycemia. Sucralose alone (at any concentration) did not affect metabolic outcomes. CONCLUSIONS:Diet sodas but not NNS in water augmented GLP-1 responses to oral glucose. Whether the trends toward higher insulin concentrations after NNS are of clinical importance remains to be determined. Our findings emphasize the need to test metabolic effects of NNS after chronic consumption. TRIAL REGISTRATION:The data for this manuscript were gathered from clinical trial #NCT01200940.