Laser acupuncture (LA) becomes more and more relevant in neonates and infants. With near-infrared spectroscopy (NIRS), a continuous and noninvasive measurement of tissue oxygenation is possible. Aim was to investigate, whether the application of LA was associated with any changes in regional cerebral oxygen saturation (rcSO2) in term and preterm neonates. The study included 20 neonates (12 males, 8 females). The Large Intestine 4 acupuncture point (LI 4, Hegu) was stimulated by a microlaser needle (10 mW, 685 nm laser needle EG GmbH, Germany) for 5 minutes, bilaterally. All neonates underwent polygraphic recording during undisturbed daytime sleep, including heart rate (HR), peripheral oxygen saturation (SpO2), and measurement of nasal flow. Using NIRS, rcSO2 was measured continuously. Cerebral fractional tissue oxygen extraction (cFTOE) was calculated. We did not observe any significant changes in SpO2 and HR values during the whole observation period. However, there was a significant decrease in rcSO2 (P = 0.003) within postintervention period, accompanied by a significant increase in cFTOE (P = 0.010) in postintervention period.