Background: Metabolic regulation of glucose can be altered by fasting periods. The aim of our study was to examine the glucose metabolism after an overnight fast (12 hours) as well as after prolonged fasting (36 hours of fasting) in non-obese people, people with obesity and people with type 2 diabetes (T2DM). Material and Methods: Glucose metabolism was assessed by a 75 g oral glucose tolerance test after 12 and 36 hours fasting, respectively. Area under the curve (AUC) was calculated according to the trapezoidal rule. Insulin sensitivity was estimated by using QUICKI and HOMA-IR. Results: In total 60 participants (age 43±16 years, 62% women) were included. Fasting levels of glucose, insulin and c-peptide were significantly lower in all cohorts after 36 hours vs. 12 hours of fasting (p<0.05). However, in non-obese people mean 2h-glucose level was significant higher after prolonged fasting 108.7±31.1 mg/dL vs. 79.4±18.4mg/dL after 12h fasting; p<0.05 with significantly lower 30 minutes insulin levels following prolonged fasting, while within the two other cohorts no change in this 2h-glucose was observed. Indices of insulin sensitivity improved in all groups after prolonged fasting. Conclusion: Our data demonstrate higher glucose excursions and a reduced early insulin response after 36 hours of fasting compared to an overnight fasting period in participants with various glucose tolerance levels. Disclosure N.J. Tripolt: None. P.N. Pferschy: None. A. Obermayer: None. M.L. Eckstein: Research Support; Self; Dexcom, Inc., Novo Nordisk A/S, Sanofi-Aventis. H. Kojzar: None. C. Sourij: None. O. Moser: Other Relationship; Self; Dexcom, Inc., Novo Nordisk A/S, Sanofi. B.M. Obermayer-Pietsch: None. R. Riedl: None. H. Sourij: Advisory Panel; Self; Amgen, AstraZeneca, Boehringer Ingelheim International GmbH, Merck Sharp & Dohme Corp., Novo Nordisk A/S, Sanofi. Research Support; Self; Boehringer Ingelheim International GmbH, Merck Sharp & Dohme Corp., Novo Nordisk A/S, Sanofi. Speaker’s Bureau; Self; Boehringer Ingelheim International GmbH, Novo Nordisk A/S, Sanofi.