The anaerobic threshold (AT) is a point during intense exercise that can be used to predict muscular fatigue. Determining the AT non-invasively helps to adjust exercise intensity and prevent overuse injuries. Near-infrared spectroscopy (NIRS) is an optical technology that can provide real-time information about muscle oxidative metabolism. The objective of this pilot study was to investigate the relationship between NIRS parameters of muscle oxygenation and traditional measures of exercise monitoring, such as heart rate and relative body oxygen consumption (VO2). Healthy adults with moderate to high fitness levels participated in an incremental exercise protocol on a stationary bicycle. NIRS parameters were compared to ventilatory VO2 using a metabolic cart. Respiratory Exchange Ratio (RER) textless 1.0 was used as a proxy for determining the AT. NIRS data were collected from the primary locomotor muscle (vastus lateralis - VL) and a control muscle (deltoid) using two wearable NIRS sensors. Heart rate data were collected by a wearable ECG sensor. The NIRS data showed a significant decline in VL muscle oxygenated hemoglobin (O2Hb) concentration (ptextless0.05) at one exercise stage after the AT was identified. Muscle O2Hb did not show a significant decrease in the deltoid at the AT. Furthermore, there were no noticeable changes in heart rate at the AT. Our results indicate that a wearable NIRS sensor can predict the AT in exercising muscles and may provide a localized measure of muscular fatigue during exercise.