Among the many features of memory, episodic memory includes the ability to separate initially similar experiences to avoid overlap at retrieval. This process, pattern separation, would enable differentiation of locations or objects in memory. Studies in humans and rodents using analogous tasks have linked behavioral performance to molecular mechanisms, suggesting its translational relevance. Pattern separation is impaired with aging, hippocampal damage, and Alzheimer’s disease, highlighting interest in strategies to enhance it. To examine the impact of acute and chronic physical activity on memory consolidation, 35 young adults (18–40 yrs) were recruited, including 24 sedentary individuals and 11 chronic athletes. Memory performance was assessed with version 7rep of the Memory Differentiation Task (MDT), designed to test discrimination of similar vs. different objects. Following the acquisition phase, 12 sedentary participants completed 25 min. of moderate exercise, while chronic athletes and remaining sedentary individuals watched a cycling race video. After 24 hs, all participants were retested. 2-way ANOVA revealed a significant effect of object type (similar vs. different), but no effect of physical activity condition (acute, chronic, control). Thus, previously reported acute exercise effects were not replicated, though results support MDT validity. High-density EEG was conducted to explore underlying neurophysiological mechanisms and their modulation by physical activity