In the present study we investigated whether one night of sleep deprivation can affect working memory (WM) performance with emotional stimuli. Twenty-five subjects were tested after one night of sleep deprivation and after one night of undisturbed sleep at home. As a second aim of the study, to evaluate the cumulative effects of sleep loss and of time-of-day changes on emotional WM ability, the subjects were tested every 4 h, from 22:00 to 10:00 hours, in four testing sessions during the sleep deprivation period (deprivation sessions: D1, D2, D3 and D4). Subjects performed the following test battery: Psychomotor Vigilance Task, 0-back task, 2-back task and an 'emotional 2-back task' with neutral, positive and negative emotional pictures selected from the International Affective Picture System. Results showed lower accuracy in the emotional WM task when the participants were sleep-deprived relative to when they had slept, suggesting the crucial role of sleep for preserving WM ability. In addition, the accuracy for the negative pictures remains stable during the sessions performed from 22:00 to 06:00 hours (D1, D2 and D3), while it drops at the D4 session, when the participants had accumulated the longest sleep debt. It is suggested that, during sleep loss, attentional and WM mechanisms may be sustained by the higher arousing characteristics of the emotional (negative) stimuli.