Purpose: Real-time measurement of dynamic brain activity during exercise can help advance our understanding of the role of exercise upon brain health and function. In exercise science, functional near infrared spectroscopy (fNIRS) has primarily been used to measure the effects of exercise intensity on hemodynamic responses in the cerebral cortex. However, the utility of fNIRS to measure discreet hemodynamic responses underlying brain activation associated with motor and cognitive function during exercise has not been systematically examined. Here, we compared brain activation associated with a motor and cognitive task at rest and during cycling exercise at different intensities. Methods: In separate sessions, 13 participants performed cycling exercise on an indoor trainer at a low, moderate and high intensity. We measured changes in oxygenated (HbO) and deoxygenated (HbR) hemoglobin from prefrontal, parietal, and motor regions of the cerebral cortex during a handgrip and working-memory task. Results: Our findings show significant brain activation (a concurrent increase in HbO and decrease in HbR) in contralateral motor cortex during the handgrip task and left prefrontal cortex during the working-memory task at rest and during exercise at low, moderate and high (motor task HbO only) intensities (P < 0.05). Moreover, brain activation during the handgrip and working-memory tasks was not significantly different at rest and during exercise (P > 0.05). Conclusions: This study shows that fNIRS can robustly measure motor and cognitive task-evoked changes in brain activation during cycling exercise comparable to rest. An implication of these new findings is that fNIRS can be used to determine real-time changes in brain function during exercise in healthy and clinical populations.