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Background: Accurate detection of cerebral oxygen saturation (rSO2) may be useful for neonatal brain injury prevention, and the normal range of rSO2 of neonates at high altitude remained unclear. Objective: To compare cerebral rSO2 and cerebral fractional tissue oxygen extraction (cFTOE) at high-altitude and low-altitude areas in healthy neonates and neonates with underlying diseases. Methods: 515 neonates from low-altitude areas and 151 from Tibet were enrolled. These neonates were assigned into the normal group, hypoxic-ischemic encephalopathy (HIE) group, and other diseases group. Near-infrared spectroscopy was used to measure rSO2 in neonates within 24 h after admission. The differences of rSO2, pulse oxygen saturation (SpO2), and cFTOE levels were compared between neonates from low- and high-altitude areas. Results: (1) The mean rSO2 and cFTOE levels in normal neonates from Tibet were 55.0 ± 6.4% and 32.6 ± 8.5%, significantly lower than those from low-altitude areas (p < 0.05). (2) At high altitude, neonates with HIE, pneumonia (p < 0.05), anemia, and congenital heart disease (p < 0.05) have higher cFTOE than healthy neonates. (3) Compared with HIE neonates from plain areas, neonates with HIE at higher altitude had lower cFTOE (p < 0.05), while neonates with heart disease in plateau areas had higher cFTOE than those in plain areas (p < 0.05). Conclusions: The rSO2 and cFTOE levels in normal neonates from high-altitude areas are lower than neonates from the low-altitude areas. Lower cFTOE is possibly because of an increase in blood flow to the brain, and this may be adversely affected by disease states which may increase the risk of brain injury. [ABSTRACT FROM AUTHOR]