Validation of a Stress Reactivity Assessment Protocol for Children Aged 4–5 Years: Exploring the Influence of Sex, Emotional Responses, and Crying

Abstract

Background: Early childhood is an important period for the development of stress regulation systems, yet validated protocols to assess stress reactivity in preschoolers remain scarce. The current study aimed to validate a stress reactivity laboratory protocol based on the matching task in a Spanish sample of 4–5-year-old children, while exploring the influence of sex, emotional responses, and potential confounding variables. Methods: Fifty-eight preschoolers participated in the Stress Reactivity Task for Preschoolers (SRTP), which included six salivary samples for the measurement of cortisol (as a marker of hypothalamic-pituitary-adrenal [HPA] axis activity) and alpha-amylase (as a marker of sympathetic nervous system [SNS] activity). Behavioral and emotional responses were also coded. Statistical analyses included repeated-measures GLMs, paired t-tests, and correlation analyses to evaluate biomarker patterns and confounders. Results: The SRTP effectively elicited a stress response: 77.6% of children were classified as alpha-amylase responders, and 64.9% as cortisol responders. Alpha-amylase levels increased sharply post-task and gradually returned to baseline within 40 minutes. In contrast, cortisol levels peaked later and remained elevated for a longer period. No correlation was found between the two biomarkers. Emotional and observational data supported the presence of stress, with significant increases in anger, sadness, and nervousness during the task. Notably, girls exhibited faster cortisol reactivity and greater sadness than boys. Among all examined variables, crying emerged as the most influential confounder, being strongly associated with heightened cortisol reactivity. Conclusions: The SRTP is a valid and sensitive protocol for assessing stress reactivity in preschool-aged children. It enables simultaneous assessment of SNS and HPA axis activity and captures meaningful interindividual differences. These findings contribute to a more nuanced understanding of early stress physiology and may inform future longitudinal studies and preventive interventions.