Adipose tissue is an endocrine organ involved in storage and release of energy, but also in regulation of energy metabolism in other organs via secretion of peptide and protein hormones (adipokines). Especially visceral adipose tissue has been implicated in the development of metabolic syndrome and type 2 diabetes. Factors secreted by the stromal-vascular fraction contribute to the secretome and modulate adipokine secretion by adipocytes. Therefore, we aimed at the characterization of the adipose tissue secretome rather than the adipocyte cell secretome. The presence of serum proteins and intracellular proteins from damaged cells, released during culture, may dramatically influence the dynamic range of the sample and thereby identification of secreted proteins. Part of the study was therefore dedicated to the influence of the culture set-up on the quality of the final sample. Visceral adipose tissue was cultured in five experimental set-ups and the quality of resulting samples was evaluated in terms of protein concentration and protein composition. The best set-up involved one wash after the first hour in culture followed by two or three additional washes within an eight hour period, starting after overnight culture. Thereafter, tissue was maintained in culture for additional 48 to 114 hrs to obtain the final sample. For the secretome experiment, explants were cultured in media containing ¹³C₆,¹⁵N₂L-lysine to validate the origin of the identified proteins (adipose tissue or serum derived). In total, 259 proteins were identified with >99% confidence. 108 proteins contained a secretion signal peptide of which 70 incorporated the label and were considered secreted by adipose tissue. These proteins were classified into five categories according to function. This is the first study on the (human) adipose tissue secretome. The results of this study contribute to a better understanding of the role of adipose tissue in whole body energy metabolism and related diseases.