Fat layer thickness (FLT) affects near infrared spectroscopy (NIRS) measurement. The aim of this study was to determine an indicator of muscle function with less influence of FLT, even without normalization, by comparing muscle O2 dynamics during exercise in subjects with similar physical characteristics except for FLT. Healthy male subjects with thick FLT (n = 5, FLT: 5.3 ± 0.4 mm) and those with thin FLT (n = 6, FLT: 3.5 ± 0.5 mm) participated in this study. All subjects performed constant work cycling exercise (CWE) at moderate intensity for 6 min. Oxygenated hemoglobin concentration, deoxygenated hemoglobin concentration and total hemoglobin concentration were monitored by using time resolved NIRS (NIRTRS). VO2peak was not significantly different between THICK and THIN (THICK: 54.0 ± 1.8, THIN: 50.0 ± 6.2 mL/kg/min). Changes in all NIRTRS variables at the onset of CWE were significantly larger in THIN than THICK, and the changes in each variable were significantly correlated to FLT. In contrast, there were no relationships between changes in NIRTRS variables during CWE and FLT. These results suggest that muscle deoxygenation at the onset of exercise, which is used as muscle O2 extraction, can be strongly influenced by FLT. In contrast, muscle oxygenation during CWE, which is used as exercise-induced functional hyperemia , may not be influenced by FLT.