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Treatment of newborns in neonatal intensive care units (NICUs) is associated with exposure to multifactorial stressors, which adversely affect the physical and psychological status of infants as well as the psycho-emotional state of their mothers. In this context, the use of non-invasive assessment of stress biomarkers has gained increasing importance. Aim. To evaluate neonatal and maternal stress in the NICU by measuring indicators of sympathoadrenal and serotonergic system function and to analyze stress-inducing factors. Materials and Methods. The study included 117 newborns admitted to the NICU of the regional perinatal center. The infants were divided into three groups according to gestational age at birth: Group 1 – extremely and very preterm infants (EVPI), <32 weeks, n = 35; Group 2 – moderate and late preterm infants (MLPI), ≥32 weeks, n = 61; Group 3 – term newborns, n = 21. The following stress biomarkers were measured: 5-hydroxyindoleacetic acid (5-HIAA), a serotonin metabolite and functional marker of the serotonergic system; and salivary alpha-amylase (sAA), a biomarker of the sympathoadrenal system activity reflecting the sympathetic division of the autonomic nervous system response to stress. Written informed consent was obtained from the parents of all participants. The study was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the Bioethics Committee of I. Ya. Horbachevsky Ternopil National Medical University, Ministry of Health of Ukraine (Protocol No. 73, April 3, 2023). Statistical analysis was conducted using STATISTICA 13.0 software (StatSoft Inc., Tulsa, OK, USA). Medians (Me), upper quartiles (Uq), and lower quartiles (Lq) were calculated. For comparison of numerical variables with non-normal distribution, the Mann-Whitney U test (for two independent groups), the Kruskal–Wallis test (for three independent groups), and the Wilcoxon signed-rank test (for two dependent groups) were applied. Statistical significance was set at p < 0.05. The research project was entitled “Assessment of neonatal and parental stress in the neonatal intensive care unit and methods of its correction”, State registration No. 0123U100063, the study period was December 2022 – November 2025. Results. In extremely preterm infants, urinary 5-HIAA levels increased after skin-to-skin contact (SSC) from 2.064 [1.423; 3.402] to 4.366 [2.592; 9.468] mg/L (p = 0.008). In the moderate and late preterm group, levels increased from 2.247 [1.530; 3.217] to 5.001 [2.812; 9.567] mg/L (p < 0.001). In term infants, 5-HIAA levels increased from 2.452 [1.861; 5.587] to 4.293 [3.565; 8.420] mg/L; however, this change was not statistically significant (p = 0.075). Decreased 5-HIAA levels in preterm infants were associated with low Apgar scores (<7 points), need for resuscitation, surfactant administration, mechanical ventilation, intraventricular hemorrhage, necrotizing enterocolitis, patent ductus arteriosus, anemia, and neonatal jaundice requiring phototherapy. In infants <32 weeks’ gestation, sAA levels after SSC decreased from 52.74 [38.54; 107.10] to 19.65 [15.15; 26.98] U/mL (p = 0.001). In infants ≥32 weeks, sAA levels decreased from 71.49 [34.98; 106.46] to 23.51 [17.39; 34.36] U/mL (p < 0.001). In term infants, sAA levels decreased from 84.24 [61.25; 101.03] to 31.37 [25.62; 42.78] U/mL (p < 0.005). Maternal sAA levels decreased correspondingly: in mothers of extremely and very preterm infants from 88.85 [51.66; 109.90] to 34.13 [23.59; 57.45] U/mL (p = 0.033); in mothers of moderate and late preterm infants from 68.85 [42.30; 98.64] to 25.03 [17.30; 29.59] U/mL (p < 0.001); and in mothers of term infants from 79.34 [54.21; 92.77] to 38.29 [22.88; 47.50] U/mL (p < 0.005). Breastfeeding in the NICU was associated with additional reduction in maternal stress: maternal sAA levels before SSC were 49.20 [28.19; 75.64] U/mL in breastfeeding mothers compared with 105.90 [80.41; 148.10] U/mL in mothers who formula-fed their infants (p = 0.046). Conclusions. Skin-to-skin contact and rooming-in in the NICU are evidence-based interventions that effectively reduce stress in both mothers and newborns, as indicated by changes in markers of serotonergic and sympathoadrenal system activity, whereas neonatal morbidity is associated with increased stress levels. Breastfeeding of preterm infants in the NICU is associated with a significant reduction in maternal stress.