Near-infrared spectroscopy is used to quantify the subcutaneous adipose tissue thickness (ATT) over five muscle groups (vastus medialis, vastus lateralis, gastrocnemius, ventral forearm and biceps brachii muscle) of healthy volunteers (n=20). The optical lipid signal (OLS) was obtained from the second derivative of broad band attenuation spectra and the lipid absorption peak (lambda=930 nm). Ultrasound and MR imaging as well as mechanical calliper readings were taken as reference methods. The data show that the OLS is a good predictor for ATT (<16 mm) with absolute and relative errors of <0.8 mm and <24%, respectively. The optical method compares favourably with calliper reading. The finding of a non-linear relationship of optical signal vs. ultrasound is explained by a theoretical two-layer model based on the diffusion approximation for the transport of photons. The crosstalk between the OLS and tissue hemoglobin concentration changes during an incremental cycling exercise was found to be small, indicating the robustness of OLS. Furthermore, the effect of ATT on spatially-resolved spectroscopy measurements is shown to decrease the calculated muscle hemoglobin concentration and to increase oxygen saturation.