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BACKGROUNDS. Pain in newborns is often difficult to recognize and therefore to treat. Changes in heartbeats per minute, respiration rate and peripheral oxygen saturation levels are most often considered to indicate pain in newborns. Near-infrared spectroscopy technology permits continuous bedside monitoring of regional tissue oxygen saturation by measuring changes in the concentration of oxygenated and deoxygenated hemoglobin. The aim of our study was to determine whether regional tissue oxygen saturation in the somatosensory cortex of the brain changes in response to painful stimulation. We hypothesized that painful stimulus (venipuncture) is associated with change in regional tissue oxygen saturation in the somatosensory cortex of the brain and change in heartbeats per minute, respiration rate and peripheral oxygen saturation levels. METHODS. In our prospective study, we consecutively included 28 newborns. The children were treated at the Department of Neonatal Care, University Medical Centre Ljubljana from December 2011 to April 2012. We measured regional tissue oxygen saturation in the somatosensory cortex of the brain, heartbeats per minute, respiration rate and peripheral oxygen saturation levels two minutes before the venipuncture, at the moment of venipuncture and two minutes after venipuncture. The results were tested using Tuckey’s range test. RESULTS. There was a statistically significant difference in regional tissue oxygen saturation in the somatosensory cortex of the brain at the moment of venipuncture compared to regional tissue oxygen saturation before venipuncture. We also found a statistically significant difference in heartbeats per minute. We found no statistically significant difference in respiration rate and peripheral oxygen saturation levels. CONCLUSIONS. Painful stimulation causes changes in the regional tissue oxygenation in the somatosensory cortex of the brain. Our results could indicate the use of near-infrared spectroscopy for recognizing acute pain in newborns.