High-intensity interval training (HIIT) improves skeletal muscle mitochondrial oxidative capacity in humans with different degrees of insulin sensitivity, whereas it increases insulin sensitivity along with number of circulating small extracellular vesicle (SEV) mainly in insulin resistant individuals with or without T2D (T2D, IR) . In order to examine the sustainability of metabolic effects induced by HIIT, this study tested whether the observed effects on insulin sensitivity, oxidative capacity and SEV number remain and/or differ between insulin sensitive (IS) , IR and T2D humans. Twenty T2D and age- and BMI-matched IR and 12 IS individuals (HbA1c in %: 7.2±0.2, 5.4±0.1, 5.4±0.1; M-value in mg*kg-1*min-1: 3.0±0.4, 4.2±0.4, 7.4±0.4) performed a 12-week HIIT cycling protocol for 3 d/week. Whole-body insulin sensitivity was assessed by hyperinsulinemic-euglycemic clamps, physical fitness (VO2max) by spiroergometry and myocellular pathways of insulin sensitivity by immunoblotting of muscle biopsies at baseline, 72 h after the last HIIT bout and 4 weeks (detraining) later. SEV were isolated from serum by size exclusion chromatography. Compared to HIIT, insulin sensitivity decreased in T2D after detraining (p<0.01) , instead VO2max decreased in T2D and IR (p<0. and p<0.05) , although it remained higher than baseline (p<0.0 and p=0.05) . Of note, HIIT-induced reduction of the nuclear factor-κB protein (p<0.vs. baseline) disappeared in IR after detraining, whereas decreased protein kinase Cε activity was sustained in T2D (p<0.vs. HIIT and p<0.vs. baseline) , who also showed higher circulating SEV number (p<0.vs. baseline) . In conclusion, HIIT-induced increases in physical fitness and SEV number, but not insulin sensitivity are maintained at least after short-term detraining in T2D. Characterization of SEV cargo might help to better understand the differences in metabolic responses to detraining in individuals with and without T2D Disclosure L.Mastrototaro: None. J.Szendroedi: Advisory Panel; Boehringer Ingelheim International GmbH. M.Roden: Advisory Panel; Eli Lilly and Company, Research Support; Boehringer Ingelheim International GmbH, Nutricia, Speaker's Bureau; Novo Nordisk. M.Apostolopoulou: n/a. N.Saatmann: None. C.Granata: None. S.Hartwig: None. Y.Karusheva: None. S.Gancheva: None. S.Lehr: None. H.Al-hasani: Consultant; Bayer AG.