Objective: To measure the effects kangaroo mother care (KMC) on the regional cerebral oxygen saturation (rSO2) measured with near-infrared spectroscopy (NIRS) in premature infants and to study the physiological stability and comfort of the infants during such interventions. Design: This was a prospective, single-centered, single-blind, 2-arm, parallel-group randomized controlled trial conducted. Settings: The study was carried out in a tertiary neonatal intensive care unit in Medical Faculty Hospital in Konya/Turkey. Participants: Premature infants born between 24 and 36+6 weeks. Participants were randomly assigned to one of two groups: kangaroo care (n = 20) and control groups (n = 20). The rSO2, oxygen saturation (%SpO2), heart rate (HR), respiratory rate, body temperature, and comfort levels of the infants were evaluated in three stages. Findings: In the 60th min measurement of the intervention group, rSO2, body temperature, heart rate, respiratory rate, and, comfort level, the comfort level was found to be higher and significant in the 30th and 60th min measurements (p < 0.001). In the intervention group, the rSO2 (p < 0.001), body temperature (p < 0.001), HR (p < 0.001), SpO2 (p < 0.001), respiratory rate (p < 0.001), and comfort levels (p < 0.001) scores with the control group and the group × time interaction was significant. Key conclusions: KMC moderately increased the rSO2 levels and also created a moderate effect size on the physiological parameters and comfort levels of the newborns, which implicates its short-term benefits for premature infants. Practice implications: KMC may be beneficial in stabilizing rSO2 and physiological parameters and increasing comfort in premature infants. The trial was registered in ClinicalTrials.gov (identifier: NCT04725435).