Hypercortisolaemia (Cushings Syndrome) is characterised by abdominal fat accumulation and gluteofemoral fat loss. The mechanisms underpinning this fat mass redistribution are unknown. Adipose tissue blood flow (ATBF) and net fatty acid release rate (lipolysis) are important determinants of depot-specific fat mass. We hypothesized that hypercortisolaemia may result in increased femoral ATBF and lipolysis promoting gluteofemoral fat mass loss. We recruited nine healthy male volunteers (median age 28 years, range 1957; BMI 26.5 kg/m2, range 22.730.4) who underwent in vivo assessment of femoral ATBF and lipolysis using radioactive Xenon washout and the arterio-venous difference technique. All subjects were studied twice, following a preceding infusion period with hydrocortisone (0.2 mg/kg per min for 16 h) and saline, respectively. Infusions were given in a randomised double-blind order. On each of the two study days ATBF and lipolysis were studied in the fasting state and following a 75 g glucose drink. Hydrocortisone infusion increased plasma cortisol significantly (AUC 2009±565 vs 77±5 nmol/l, P=0.012). Compared to saline, hydrocortisone increased systemic plasma non-esterified fatty acids (NEFA) by 62±13% during the fasting state (AUC 944±53 vs 597±39 μmol/l, P=0.008). Expectedly, glucose suppressed systemic lipolysis, although postprandial NEFA remained higher after hydrocortisone (AUC 341±23 vs 178±11 μmol/l, P=0.008). Hydrocortisone infusion induced a steady increase in femoral ATBF during the postabsorptive phase (AUC 4.4±0.6 vs 2.4±0.4 ml/min×100 g/tissue, ANOVA P=0.009 infusion type×time). Following oral glucose load, femoral ATBF increased further compared to saline (AUC 7.9±1.4 vs 3.3±0.5 ml/min×100 g/tissue, P=0.011). During control conditions, fasting femoral NEFA release rate was high (AUC 671±232 nmol/min×100 g/tissue), and was inhibited following glucose (AUC 413±162 nmol/min×100 g/tissue). Hydrocortisone did not have any effect on femoral fasting or postprandial lipolysis (P=0.285 and P=0.447 compared to saline, respectively). Hypercortisolaemia increases femoral ATBF, but not lipolysis, suggesting differential glucocorticoid effects in the vasculature and adipocytes in vivo. Further research should focus on the effect of hypercortisolaemia on depot-specific fatty acid uptake.