Although night-shift work (NSW) is associated with a higher risk for several physical and mental disorders, the impact of NSW on pain perception is still unclear. This study investigates the impact of NSW on cold pain perception considering the impact of mood and sleepiness. Quantitative sensory testing (QST) was performed in healthy night-shift workers. Cold pain threshold as well as tonic cold pain was assessed after one habitual night (T1), after a 12-hour NSW (T2) and after one recovery night (T3). Sleep quality was measured with the Pittsburgh Sleep Quality Index (PSQI) before T1, sleepiness with the Stanford Sleepiness Scale (SSS) and mood with a German short-version of the Profile of Mood States (ASTS) at T1, T2 and T3. Depending on the distribution of the data, ANOVAs or Friedman tests as well as t- or Wilcoxon tests were performed. Nineteen healthy shift-workers (13 females; 29.7 ± 7.5 years old; 8.1 ± 6.6 years in shift work, PSQI: 4.7 ± 2.2) were included. Tonic cold pain showed a significant difference between T1 (48.2 ± 27.5 mm), T2 (61.7 ± 26.6 mm; effect size: Cohen's d=.49; percent change 28%), and T3 (52.1 ± 28.7 mm) on a 0-100 mm Visual Analog Scale (p = 0.007). Cold pain threshold changed from 11.0 ± 7.9 °C (T1) to 14.5 ± 8.8 °C (T2) (p = 0.04), however, an ANOVA comparing T1, T2, and T3 was not significant (p = 0.095). Sleepiness (SSS) and mood (ASTS) changed significantly between T1, T2 and T3 (p-values < 0.01). The change of mood but not of sleepiness correlated with the difference in tonic cold pain from T1 to T2 (R: 0.53; R2: 0.29; p = 0.022). NSW increases cold pain perception. The same tonic cold pain stimulus is rated 28% more painful after NSW and normalizes after a recovery night. Increases in cold pain perception due to NSW appear to be more strongly related to changes in mood as compared to changes in sleepiness.