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Objectives To investigate the emergence of biological rhythms in the first months of life in human infants, by measuring age-related changes in core body temperature during night-time sleep, hormones (cortisol and 6-sulfatoxymelatonin) and the expression of a clock-controlled gene H3f3b in oral epithelial cells. Design Observational longitudinal study. Setting We measured overnight core body temperature, actigraphy, day-night urinary cortisol and 6- sulfatoxymelatonin, as well as circadian gene expression, in infants at home from March 2007 to July 2008 in Leicester. Participants We recruited 35 healthy Caucasian infants who were born at term. They were monitored from 6 to 18 weeks of age. Results At 8 weeks of age the day-night rhythm of cortisol secretion was the first to appear followed by 6-sulfatoxymelatonin 1 week later; at the same time that night-time sleep was established. At 10 weeks, the maximum fall in deep body temperature occurred with the onset of night-time sleep, followed at 11 weeks by the rhythmical expression of the H3f3b gene. Conclusions In human infants, there is a clear sequential pattern for the emergence of diurnal biological rhythms between 6 and 18 weeks of postnatal age, led by the secretion of cortisol and linked with the establishment of consolidated night-time sleep. It is likely that this represents part of a maturation and adaption process as infants gain equilibrium with their external environment after birth. [ABSTRACT FROM AUTHOR]