OBJECTIVE: Disease and hospitalization generate stress, which can affect the response to treatment. Humor has been used in many hospitals to decrease stress. The aim of this study was to evaluate the impact of a humor therapy program on stress levels in pediatric inpatients. METHODS: In the first phase, an intervention and a control group were studied over 2 consecutive 3-month periods; the interventions were performed by a team of artists trained in humor therapy. Salivary cortisol levels were measured by enzyme-linked immunosorbent assay, and the Weisz test, a pictorial chart that determines subjective stress perception, and the Parker test, which assesses objective stress, were applied. In the second phase, salivary cortisol levels were measured and the Weisz test was administered before and after the interventions. RESULTS: A total of 306 patients were recruited into this study: 198 in the first phase (94 in the intervention group and 104 in the nonintervention group) and 108 in the second phase. There were no differences between groups regarding age, sex, or medical diagnosis. The children in the intervention group presented lower cortisol levels, lower scores on the Parker test, and higher scores on the Weisz test than children in the nonintervention group. In the second phase, the children showed lower salivary cortisol levels and higher scores on the Weisz test after the intervention. CONCLUSIONS: Humor therapy has beneficial effects on stress and cortisol levels in pediatric inpatients. This supports the implementation and reinforcement of these therapies in pediatric hospitals.