Objective. Near-infrared spectroscopy (NIRS) measurements of tissue oxygen saturation (StO2) are useful for the assessment of skeletal muscle perfusion and function during exercise, however, they are influenced by overlying skin and adipose tissue. This study explored the extent and nature of the influence of adipose tissue thickness (ATT) on StO2. Approach. NIR spatially resolved spectroscopy (SRS) derived oxygenation was measured on vastus lateralis in 56 patients with chronic heart failure (CHF) and 20 healthy control (HC) subjects during rest and moderate intensity exercise with simultaneous assessment of oxygen uptake kinetics ($τ$ ). In vitro measurements were performed on a flow cell with a blood mixture with full oxygen saturation (100%), which was gradually decreased to 0% by adding sodium metabisulfite. Experiments were repeated with 2 mm increments of porcine fat layer between the NIRS device and flow cell up to 14 mm. Main results. Lower ATT, higher $τ$ , and CHF were independently associated with lower in vivo StO2 in multiple regression analysis, whereas age and gender showed no independent relationship. With greater ATT, in vitro StO2 was reduced from 100% to 74% for fully oxygenated blood and increased from 0% to 68% for deoxygenated blood. Significance. This study shows that ATT independently confounds NIR-SRS derived StO2 by overestimating actual skeletal muscle oxygenation and by decreasing its sensitivity for deoxygenation. Because physiological properties (e.g. presence of disease and slowing of $τ$ ) also influence NIR-SRS, a correction based on optical properties is needed to interpret calculated values as absolute StO2.