Objective <p>Laughter is recognized for its potential to alleviate stress. Salivary α-amylase (sAA) is a well-known stress marker; however, the relationship between salivary oxytocin (sOXT) and stress reducing effects by laughter remains unclear. We previously determined the relationship between laughter and optimism, anxiety, and amylase; In this study, the relationship between spontaneous laughter and stress reduction during a live comedy performance (LCP) was investigated by measuring sAA and sOXT activity before and after the performance.</p> Results <p>Pre-LCP sAA was significantly positively correlated with a decreasing post-sAA/pre-sAA ratio; the scale of this effect was age dependent. Furthermore, participants who reported laughing ‘several times a month’ showed the highest value in the post-/pre-LCP sAA ratio. In contrast, those who reported laughing ‘many times a day’ had the lowest value. Contrary to our expectations, sOXT levels were significantly lower in post-LCP samples than they were in pre-LCP samples. This is thought to be because OXT has an anti-stress hormone effect, and stress was reduced after LCP, resulting in a decrease in oxytocin secretion. Our findings offer new insight into the stress-reducing effects of laughter.</p>