Neuroglobin (Ngb), a protein located in the mammal's brain, is involved in oxygen transport and free radical scavenging inside the neurons. Ngb colocalizes with choline acetyltransferase in the laterodorsal tegmental nucleus and in the pontine tegmental nucleus, both involved in the sleep-wake cycle regulation. Some studies have shown that free radicals accumulated during prolonged wakefulness are removed during sleep. Therefore, Ngb could act as a regulator of free radicals generated during prolonged wakefulness in the brain. The aim of this study was to determine whether prolonged wakefulness affects Ngb immunoreactivity because of increases in the oxidative stress induced by continuous neuronal activity. For this purpose, male adult Wistar rats were implanted with electrodes for sleep recordings and were divided into control and sleep-deprived groups. Sleep deprivation was carried out for 24 h by gentle handling of the animals. Sleep-wake activity was determined during the deprivation period or 24 h of control conditions. Subsequently, both groups of animals were killed and their brains were obtained and processed for Ngb immunohistochemical analysis and detection of lipid peroxidation. Our data found no evidence of increased oxidative stress in the brains of sleep-deprived animals compared with the controls. The number of Ngb-positive cells was decreased in the sleep-deprived animals in all analyzed areas of the brain compared with the control group. Our results suggest that Ngb could be involved in sleep regulation, independent of its role in the control of oxidative stress.