AbstractAutonomous cortisol secretion (ACS) of an adrenal incidentaloma (AI) is associated with mild cortisol excess that could result in poor metabolic and cardiovascular outcomes. The biological activity of glucocorticoids depends on the unbound, free fraction. We aimed to evaluate plasma free cortisol (FC) concentrations in patients with ACS in this cross-sectional study. One hundred and ten AI patients in 3 groups; non-functioning (NFA, n=33), possible ACS (n=65), ACS (n=12) were enrolled. Following measurements were conducted: Clinical data and total serum cortisol (TC), plasma corticotrophin (ACTH), serum dehydroepiandrosterone sulfate (DHEA-S), cortisol after 1 mg dexamethasone by both immunoassay and LC-MS/MS (DexF), serum corticosteroid binding globulin (CBG), plasma dexamethasone concentration [DEX] and plasma FC by LC-MS/MS. Patients with ACS featured an unfavorable metabolic profile. Plasma [DEX] and serum CBG levels were similar between groups. Plasma FC was significantly higher in ACS when compared to NFA and possible ACS groups p<0.05 and p<0.01, respectively. In multiple regression analysis DexF (beta=0.402, p<0.001) and CBG (beta=−0.257, p=0.03) remained as the independent predictors of plasma FC while age, sex, BMI, smoking habit, and existing cardiovascular disease did not make a significant contribution to the regression model. In conclusion, the magnitude of cortisol excess in ACS could lead to increased plasma FC concentrations. Further studies in AI patients are needed to demonstrate whether any alterations of cortisol affinity for CBG exist and to establish whether plasma FC concentrations predict the unfavorable metabolic profile in ACS.