The increasing use of robotic devices in clinical settings for rehabilitation and assistance underscores the need to understand their effects on muscle activation patterns. Prior studies have suggested that excessive assistance from robotic devices reduces voluntary control, leading to potential negative consequences on rehabilitation outcomes. However, the observation of muscle activation during exoskeleton-assisted walking in unimpaired individuals suggests the absence of adaptive responses to short-term exposure at high levels of assistance. The objective of this study is to determine whether prolonged exposure to maximum exoskeleton assistance induces adaptive changes in muscle activity and to analyze if distinct muscle activation profiles emerged during assisted versus unassisted walking. To achieve this, we performed the electromyographic analysis of eight bilateral lower limb muscles in ten participants during a one-hour training session. The results revealed a decrease in muscle activity over time. Furthermore, assisted walking exhibited distinct muscle patterns compared to unassisted walking, demonstrating that the level of assistance, along with the exoskeleton’s structure, significantly influences muscle activity. These findings hold significance for optimizing assistance regulation in exoskeleton-assisted walking, in terms of levels and timing of assistance changes, to enhance rehabilitative outcomes. Understanding how exoskeletons influence muscle activation can lead to improved rehabilitation strategies, maximizing the benefits of this technology for enhancing walking ability in people with neurological conditions or injuries.