To examine whether laughter yoga (LY), i.e., simulated laughter, alters cortisol and dehydroepiandrosterone (DHEA) levels and cortisol/DHEA (C/D) ratios.In a randomized controlled trial, 120 healthy university students were allocated to experiencing LY, watching a comedy movie (spontaneous laughter), or reading a book. Salivary cortisol and DHEA levels were measured immediately before, immediately after, and 30 min after the intervention.Cortisol levels and C/D ratios significantly decreased by time in the LY and comedy movie groups. Significant group*time interactions were found between these two groups for cortisol levels and C/D ratios. DHEA levels did not change by time in the LY group.LY decreased cortisol levels and C/D ratios but did not affect DHEA levels. Simulated and spontaneous laughter differently affected the dynamics of cortisol levels and C/D ratios. Effect of spontaneous laughter on the cortisol dynamics lasted longer than that of simulated laughter. (UMIN000019409).