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Overground walking powered exoskeletons (OPE) assist the lower limbs by initiating and replicating step kinematics while supporting the trunk, enabling users to maximize movement repetitions for motor learning. Although the benefits of this technology have been demonstrated in a context of rehabilitation, its effectiveness varies among individuals. Understanding the neurophysiological effects of OPE-assistance in non-disabled individuals is crucial before applying this research in persons with central nervous system (CNS) lesion. This exploratory study aims to describe and compare the effects of two levels of robotic assistance on muscle activation and fatigue during overground walking in non-disabled individuals. Participants took part in two walking sessions (familiarization and assessment) with an OPE. During the assessment, they completed 3 walking trials (300 m each). In the first and the third trials, 100% assistance was provided by the OPE, while 40% assistance was used in the second trial. After each trial, participants assessed their perceived workload using the NASA-TLX questionnaire. Electromyographic data were collected from key lower-limb muscles involved in walking. The mean amplitude of muscle activity in specific zones, the median frequency of the power spectrum, and the NASA-TLX score were calculated for each trial and compared across trials using a Friedman test. A decrease in medial hamstrings’ activation was noted when participants transitioned from full assistance to 40%. These modifications persisted upon a second exposure to 100%. While no significant differences across trials were observed in the medial gastrocnemius muscle, OPE-assisted walking led to an extinction of rectus femoris and tibialis anterior activity. No muscular fatigue was induced by walking with the OPE, regardless of the assistance level. Perceived workload significantly decreased between the first trial and the subsequent two, particularly in terms of mental demands and effort. Lower-limb muscle activity was more influenced by walking with OPE-assistance than by the level of assistance itself. The present study highlights the importance of providing more familiarization sessions. As these results reflect the effects observed in non-disabled individuals, further experiments are needed to investigate muscle activation and fatigue in response to varying assistance levels in individuals with a CNS lesion.