Aim: Type II diabetes and obesity are associated with an enhanced release of a number of adipocytokines. Hyperinsulinemia - frequently present in type II diabetes and obesity - might be one of the drivers of the enhanced production of adipocytokines. The aim of this study was to investigate the interstitial levels of cytokines in subcutaneous adipose tissue (SCAT) in response to hyperinsulinemia and the effect of weight-reducing hypocaloric diet on this regulation in obese subjects. Methods: 13 obese pre-menopausal women participated in the study. Concentrations of 7 cytokines were measured in plasma and in AT interstitial fluid collected by microdialysis method during euglycemic-hyperinsulinemic clamp and during control infusion of physiological saline. A subgroup of 6 women underwent a 4-week very-low-calorie diet (VLCD). Microdialysis during the clamp was performed before and at the end of VLCD. Results: Hyperinsulinemia induced an increase of monocyte chemoatractant protein (MCP-1) and IL-6 SCAT interstitial and plasma levels, and elevated IL-8 levels in SCAT. The relative changes of IL-6 levels in the dialysate correlated with changes of IL-8 and MCP-1. The interstitial and plasma levels of IL-1β, IL-10, TNFα and plasminogen activator inhibitor (PAI-1) remained unchanged in response to hyperinsulinemia. VLCD resulted in an enhancement of the hyperinsulinemia-induced augmentation of MCP-1, IL-6 and IL-8 interstitial levels. Conclusion: Hyperinsulinemia upregulates the interstitial levels of MCP 1, IL-6 and IL-8 in SCAT in obese women while it does not affect IL-1β, IL-10, TNFα and PAI-1 levels. Hypocaloric diet associated with weight reduction enhances the hyperinsulinemia-induced up-regulation of MCP 1, IL-6 and IL-8 in SCAT.