| Abstract: | The aim of the present study was to evaluate the development of the circadian rhythm of the salivary cortisol in premature infants and its correlation with the onset of the sleep–activity behavior pattern during the first 3 weeks of life under controlled light:dark conditions. Furthermore, we investigated the influence of acoustic stimulation by audiotaped lullabies or the maternal voice on the cortisol values and long-term sleep–activity patterns. The study was a block-randomized, prospective clinical trial with a study population of 62 preterm neonates (30?N?=?20; maternal voice: N?=?20) with a matched control group (N?=?22). The acoustic stimulation took place every evening between 20:00 and 21:00?h for 30?min over a period of 2 weeks. The cortisol values and activity–rest behavior of the neonates were determined during the first 3 weeks of life on the 1st, 7th and 14th day. Actigraphic monitoring was used to record the activity pattern continuously over 24?h and a validated algorithm for neonates was used to estimate sleep and wakefulness. The saliva samples were obtained 10?min before and 10?min after the acoustic interventions for the study groups. Additionally, saliva samples were obtained from the control group seven times over a 24-h period (20:00, 21:00, 01:00, 05:00, 08:00, 13:00 and 17:00?h). The cortisol data were analyzed by fast Fourier transformation to assess periodic characteristics and frequencies. Hierarchical linear modeling was further performed for the statistical analysis. Results: The cortisol rhythm analysis indicated a circadian rhythm pattern for only one premature infant, all others of the neonates showed no circadian or ultradian rhythm in cortisol. Cortisol level of the premature neonates was significantly higher during the first day of the study period at night-time (median: 17.1?nmol/L, IQR?=?9.7–24.4?nmol/L) than on days 7 (median: 9.6?nmol/L, IQR?=?4.7–14.6?nmol/L; Tukey-HSD, z?=?4.12, p?z?=?2.89, p?z?=?2.50, p?z?=?2.51, p?
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