Adipose tissue (AT) emerges as an endocrine organ and a key regulator of the metabolically triggered inflammation. The aims of this study were: 1) to investigate the usefulness of a multiplexed bioassay at characterizing a panel of adipokines in subcutaneous (sc) microdialysate samples; and 2) to determine whether lean and obese individuals differ in their interstitial adipokines levels following microdialysis (MD) probe insertion. Ultrafiltrating MD membranes were inserted in opposite sites of the sc abdominal AT of six lean (L) and six obese (OB) males at the beginning (M1) and during the last 120 min (M2) of the study. Interstitial (i) and serum (s) concentrations of adipokines were quantified by Luminex technique and ELISA at 60-min intervals for 5 h. In comparison with L, OB subjects exhibited elevated (i)leptin (p<0.001), (i)IL-8 (p<0.05) and (i)IL-18 levels (p=0.05), as well as higher serum concentrations of leptin (p<0.0001), (s)IL-6 (p<0.0001), (s)TNF- (p<0.001), (s)IL-8 (p=0.01) and (s)IP-10 (p<0.05). In samples from the M1 membranes, leptin decreased, IL-1, IL-18 and RANTES remained relatively stable, while IL-6, IL-8 and MCP-1 significantly increased after the first hour (p<0.0001 vs basal). Notably, either the magnitude of increase from the initial values or the time pattern of all the adipokines in M1 and M2 dialysates were similar between the groups. In conclusion, the current work provides valuable information on the optimal time frame to collect in situ AT microdialysate samples. Further studies are however needed to unravel the intricate interplay of cytokines in AT interstitial fluid.