Background: It is well known that the metabolic regulation of glucose can be influenced by dietary interventions, including alterations in meal timing. Aim of our analysis was to examine the glucose metabolism after an overnight fast (10 hours) and after prolonged fasting (36 hours of fasting). Material and Methods: Glucose metabolism was assessed by a 75 g oral glucose tolerance test after an overnight fasting period of 10 hours and a fasting period of 36h. Beta-cell function was quantified by 1st and 2nd phase insulin secretion indices. Results: We included 37 healthy subjects (mean age 48±10 years; 54% women) with a mean body mass index (BMI) of 25.2±3.4 kg/m2. After prolonged fasting mean 2h-glucose and AUC for glucose value were significantly higher than after 10h fasting (2h-glucose: 110±27 mg/dL vs. 89±24 mg/dL; p<0.001) (AUCglucose: 5336±3302 mg/dL/min vs. 3692±2266 mg/dL/min; p=0.002). First (745±500 vs. 982±573; p=0.012) and second phase insulin secretion (211 ± 115 vs. 266 ± 132; p=0.011) were significantly lower after 36h of fasting as compared to an overnight fast. Estimates for insulin sensitivity are similar after 10 and 36 hours of fasting. Conclusion: Our data demonstrate higher glucose excursions and a reduced insulin response after a 36 hour fasting period as compared to an overnight fasting period in healthy subjects.After 10h fastingAfter 36h fastingp-valueFasting c-peptide (ng/mL)1.39 ± 0.651.09 ± 1.030.025Fasting insulin (mU/L)7.9 ± 5.15.7 ± 5.80.042Fasting glucose (mg/dL)88 ± 988 ± 180.830Insulin after 60min (mU/L)87.6 ± 85.853.4 ± 51.80.0361st phase insulin secretion982 ± 573745 ± 5000.012HOMA-IR1.75 ±  1.181.39 ± 2.070.303Stumvoll index (ISI)0.10 ± 0.030.09 ± 0.03 0.230 Disclosure N.J. Tripolt: None. S. Stekovic: None. J. Url: None. F. Aberer: None. P.N. Pferschy: None. R. Riedl: None. B.M. Obermayer-Pietsch: None. T.R. Pieber: Consultant; Self; Arecor, AstraZeneca, Eli Lilly and Company, Novo Nordisk A/S, Sanofi. Employee; Self; CBmed. Research Support; Self; Novo Nordisk A/S, AstraZeneca. F. Madeo: None. H. Sourij: Speaker's Bureau; Self; Boehringer Ingelheim GmbH, Novo Nordisk A/S, Amgen Inc., Sanofi, MSD K.K.. Research Support; Self; AstraZeneca, Boehringer Ingelheim GmbH, MSD K.K., GI Dynamics Inc..