Purpose <p>Mental health disorders represent a critical challenge in modern society, necessitating the establishment of effective management strategies. Low-intensity exercise is thought to improve mood by activating endocannabinoids (eCBs), but the underlying mechanisms remain unclear. This study aimed to test the hypothesis that an increase in circulating eCB levels during low-intensity exercise contributes to a reduction in negative affect.</p> Methods <p>In a randomized crossover trial, 30 healthy participants completed two 60-min cycling exercise sessions at two intensities: low (30% heart rate reserve [HRR]) and moderate (50% HRR). Plasma concentrations of N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG) were measured at four points: pre-exercise (baseline), during exercise (10 and 30&#xa0;min), and immediately post-exercise (60&#xa0;min). Mood was assessed before and after each session using the Physical Activity Affect Scale.</p> Results <p>Plasma eCB concentrations did not change uniformly and showed substantial inter-individual variability in both conditions. Although low-intensity exercise significantly reduced negative affect, this change was not significantly correlated with the cumulative plasma AEA and 2-AG during the exercise. Notably, a repeated-measures correlation (rmcorr) revealed a significant negative correlation between plasma AEA concentrations and negative affect across pre- and post-exercise time points in the low-intensity condition.</p> Conclusion <p>Low-intensity exercise improved negative affect despite inconsistent aggregate changes in eCBs, specifically AEA and 2-AG. Dynamic intra-individual changes in plasma AEA, rather than cumulative concentrations, appear to drive this mental health benefit. These findings suggest a novel mechanism implicating exercise-induced AEA dynamics in mental health improvement.</p> Clinical trial registration <p> UMIN-CTR UMIN000060566, registered 4 February 2026, retrospectively registered.</p>