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Background: Clowning has been used in many hospitals, particularly for children. Studies suggest the effectiveness of this methodology, but more evidence is needed. The aim of this study was to evaluate the impact of a humour therapy intervention on biological markers, pain and anxiety levels in paediatric patients., Methods: Three different clinical contexts were chosen to assess the effect of clowning interventions: patients who were subjected to venepuncture (group 1), patients undergoing general anesthesia for any cause (group 2)and patients hospitalized in the pediatric ward without distinction of their disease (group 3). Groups 1 and 2 were divided into control (C) and intervention (I) subgroups. A saliva sample was taken from all the children to measure oxytocin and cortisol levels by ELISAs. Validated scales and crying time were used to determine pain, stress, and anxiety levels. Children in group 3 were assessed before and after the intervention, employing the same methods., Results: A total of 272 patients were included. The children in group 1 (n = 125) were 7.7 ± 3.2 years old, and 53.6% were females. 48% were in the I group, which showed decreased cortisol levels and increased oxytocin levels. The I group exhibited a decrease in perceived pain and crying time. The children in group 2 (n = 69) were aged 7.1 ± 3.5 years, and 36% were females. 51% were in the I group, which showed increased oxytocin levels and decreased cortisol levels, acute stress levels, perceived pain, and crying time. The children in group 3 (n = 78) were 8.6 ± 3.3 years old, and 54% of the children were females. There was an increase in oxytocin levels and a decrease in cortisol levels, stress levels and perceived pain following the intervention., Conclusions: This study suggested that an intervention based on clowning is an effective strategy for decreasing pain, stress, and anxiety levels in paediatric patients in different clinical contexts. These findings support the implementation of humour therapy programs in paediatric units., Competing Interests: Declarations Ethics approval and consent to participate This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Universidad Tecnológica de Pereira (Code 32–160421). Written informed consent was obtained from the parents or proxies. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)