Background/Aims: Salivary cortisol has been widely used to assess childhood stress. Yet, there is no consensus on reference concentrations, awakening response, guideline compliance and contribution of sampling factors to the variation in children's salivary cortisol levels. Methods: Samples were collected from 444 Belgian children participating in the ChiBS study (5-11 years old) on two consecutive weekdays at four moments: awakening, 30 min later, 60 min later and in the evening. A checklist requested awakening time, collection hours and guideline compliance. Results: Reference values were determined. Mixed model analyses revealed that age, time compliance and awakening time contributed significantly to the variance in cortisol levels. In only 52.5% of the children a cortisol morning increase was observed. Participants with no morning increase showed higher awakening but lower post-awakening concentrations on that day, and the morning response showed a small negative correlation with the time lag between first and second sampling. Conclusion: This study emphasizes the importance of excluding extreme time deviation and correcting for age and awakening time. Appearance of a cortisol morning increase was only found in approximately half of the children, suggesting the absence of the cortisol awakening response as a general characteristic. Also, this could partially be explained by poor time compliance.

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