Chronic pain patients often suffer from insomnia or impaired sleep which has been associated with increased pain sensitivity, but a limited amount of studies have investigated the effects of total sleep deprivation on central pain mechanisms. Therefore, the aim of this study was to determine the effects of total sleep deprivation on temporal summation, conditioned pain modulation, thermal and pressure pain sensitivity in healthy participants. Twenty-four healthy participants took part in this two-session trial. The measurements were conducted after a night of habitual sleep (baseline) and following 24 hours of total sleep deprivation. Detection thresholds for cold and warmth and pain thresholds for cold and heat were assessed. Cuff induced pressure pain detection and tolerance thresholds, temporal summation and conditioned pain modulation were assessed with user-independent, computer-controlled cuff algometry. Conditioned pain modulation was significantly impaired, temporal summation was significantly facilitated and pain sensitivity to pressure and cold pain were significantly increased at follow-up compared with baseline. In conclusion, this study found that one night of total sleep deprivation impaired descending pain pathways, facilitated spinal excitability and sensitized peripheral pathways to cold and pressure pain. Future studies are encouraged to investigate if sleep therapy might normalize pain sensitivity in sleep-deprived chronic pain patients.