Force and effort are important components of a motor task that can impact rehabilitation effectiveness. However, few studies have evaluated the impact of these factors on cortical activation during gait. The purpose of the study was to investigate the relation between cortical activation and effort required during exoskeleton-mediated gait at different levels of physical assistance in healthy individuals. Twenty-four healthy participants walked 10 m with an exoskeleton that provided four levels of assistance: 100%, 50%, 0%, and 25% resistance. Functional near-infrared spectroscopy (fNIRS) was used to measure cerebral flow dynamics with a 20-channel (plus two reference channels) device that covered most cortical motor regions bilaterally. We measured changes in oxyhemoglobin (HbO2) and deoxyhemoglobin (HbR). According to HbO2 levels, cortical activation only differed slightly between the assisted conditions and rest. In contrast, bilateral and widespread cortical activation occurred during the two unassisted conditions (somatosensory, somatosensory association, primary motor, premotor, and supplementary motor cortices). A similar pattern was seen for HbR levels, with a smaller number of significant channels than for HbO2. These results confirmed the hypothesis that there is a relation between cortical activation and level of effort during gait. This finding should help to optimize neurological rehabilitation strategies to drive neuroplasticity.