Sleep deprivation (SD) negatively affects various cognitive performances, but surprisingly evidence about a specific impact of sleep loss on subjective evaluation of emotional stimuli remains sparse. In the present study, we assessed the effect of SD on the emotional rating of standardized visual stimuli selected from the International Affective Picture System. Forty university students were assigned to the sleep group (n =20), tested before and after one night of undisturbed sleep at home, or to the deprivation group, tested before and after one night of total SD. One-hundred and eighty pictures (90 test, 90 retest) were selected and categorized as pleasant, neutral and unpleasant. Participants were asked to judge their emotional reactions while viewing pictures by means of the Self-Assessment Manikin. Subjective mood ratings were also obtained by means of Visual Analog Scales. No significant effect of SD was observed on the evaluation of pleasant and unpleasant stimuli. On the contrary, SD subjects perceived the neutral pictures more negatively and showed an increase of negative mood and a decrease of subjective alertness compared to non-deprived subjects. Finally, an analysis of covariance on mean valence ratings of neutral pictures using negative mood as covariate confirmed the effect of SD. Our results indicate that sleep is involved in regulating emotional evaluation. The emotional labeling of neutral stimuli biased toward negative responses was not mediated by the increase of negative mood. This effect can be interpreted as an adaptive reaction supporting the “better safe than sorry” principle. It may also have applied implications for healthcare workers, military and law-enforcement personnel.