Insulin's action to stimulate glucose utilization is determined by the insulin concentration in interstitial fluid (ISF) of insulin-sensitive tissues. The concentration of interstitial insulin has been measured in human subcutaneous adipose tissue and skeletal muscle, however, never in parallel. The aim of this study was to compare interstitial insulin levels between both tissue beds by simultaneous measurements, and to verify and quantify low peripheral ISF insulin fractions as found during moderate hyperinsulinemia. Nine healthy subjects (27.2 ± 0.8 yrs) were investigated. An euglycemic-hyperinsulinemic clamp was started with a primed-constant i.v. insulin infusion of 1mU kg^-1 min^-1. For a direct access to ISF macroscopically perforated open-flow microperfusion catheters were inserted into both tissues. During steady-state conditions (9.5 hours) interstitial effluents were collected in 30min fractions using five different insulin concentrations in the inflowing perfusates ('No Net Flux' protocol). Regression analysis of insulin concentrations in perfusates and effluents yielded the relative recovery and the perfusate insulin concentration, which was in equilibrium with the surrounding tissue. Thus, in s.c. adipose tissue and skeletal muscle the mean ISF to serum insulin level was calculated as 21.0 % (95% CI 17.5 - 24.5) and 26.0 % (95% CI 19.1 - 32.8; p=0.14), respectively. Recoveries for insulin averaged 51 % and 64 %, respectively. The data suggest that the concentrations of insulin arising in healthy subjects at the level of ISF per se are comparable between s.c. adipose and skeletal muscle tissue. The low interstitial insulin fractions seem to confirm reports of low peripheral insulin levels during moderate insulin clamps.