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Background. The cranial osteopathic manipulative medicine has been shown to alter regional cerebral tissue oxygenation (cStO2) in adult patients; however, there are no reports regarding the neonatal population. Objectives. To assess the influence of compression of the 4th ventricle (CV4) osteopathic procedure on cStO2 in neonates. Material and methods. Thirty-one patients born between 25 and 39 weeks of gestation were screened for inclusion in the neonatal unit. Twenty-two infants presenting with hyperstimulation of autonomous nervous system (ANS) according to the Neonatal Behavioral Assessment Scale were enrolled in the study. Near-infrared spectroscopy was used for continuous cStO2 monitoring; pulse oximeter oxygen saturation (SpO2) and heart rate (HR) measured with pulse oximetry were simultaneously monitored 10 min before CV4, during the therapy and 10 min after it was stopped. Results. Patients' condition remained stable throughout the study. There were no significant differences in the mean cStO2 values recorded before (69 ±8%), during (69 ±8%) and after CV4 (70 ±8%; p > 0.05). Mean SpO2 was almost constant during the study (96 ±4% before, 95 ±3% during and 95 ±4% after the intervention). Heart rate was also stable pre-, during and post-therapy (153 ±21 min, 151 ±18 min and 151 ±20/min, respectively). Conclusions. Compression of the 4th ventricle osteopathic procedure does not influence the cStO2 in newborns. This method seems to be well-tolerated but its clinical efficacy needs to be further investigated in this group of patients. [ABSTRACT FROM AUTHOR]