The purpose of this study was to compare the thigh muscle oxygenation state of competitive road cyclists and non-cyclists during varied pedaling frequency cycling. Six male college road cyclists (CY group) and five male students (NC group) performed four sets of cycling bouts, consisting of 2 minutes of warm up (60 rpm, 50 watts) followed by 5 minutes of pedaling (150 watts) using an electro-magnetic braked cycle ergometer at 40, 60, 90, and 120 rpm. Oxygenated hemoglobin and/or myoglobin (Oxy-Hb/Mb) and deoxygenated Hb/Mb (Deoxy-Hb/Mb) concentrations in the vastus lateralis were measured by near infrared spatially resolved spectroscopy. The Oxy-Hb/Mb level was significantly higher in the CY group than the NC group. But there was no significant intraction effect of the group and pedaling rate on the Oxy-Hb/Mb level. These results suggest that the changes in muscle oxygenation state according to pedaling cadence do not differ between cyclists and non-cyclists. And though the whole body work efficiency decreased according to increasing pedaling cadence, Oxy-Hb/Mb and Deoxy-Hb/Mb levels in the vastus lateralis remained unchanged up to 90 rpm. However, at 120 rpm, the Oxy-Hb/Mb level decreased remarkably and the Deoxy-Hb/Mb level increased. These results suggest that deoxygenation in the vastus lateralis at 120 rpm was higher than that for lower frequencies. And, conversely, oxygen uptake in the vastus lateralis might have increased steeply at 120 rpm. It may be that the maximum pedaling cadence that would not reduce work efficiency in the vastus lateralis is around 90 rpm.