Background And Objective: Physicians may experience periods of acute sleep deprivation while on-call, in addition to baseline chronic sleep deprivation which may affect physicians′ performance and patients′ safety. The purpose of this study was to determine the effect of acute sleep deprivation due to working long on-call shifts on mood and alertness, both of which may impair physicians′ performance. Methods: Eighty-eight junior physicians working in one university hospital completed a questionnaire, before and after completion of a shift, that collected data regarding socio-demographic factors, patterns of work and sleep, Profile of Mood States (POMS), and Stanford Sleepiness Scale. Based on duration of sleep the physicians had during on-call in comparison to their usual average sleep, the participants were categorized into group 1 (those who slept many fewer hours), group 2 (those who slept fewer hours), or group 3 (those who slept the same number of hours). Results: More than 87% of the participant slept 5 or fewer hours while working an on-call shift. Among all participants, the percentage of physicians who were alert post-on-call was significantly reduced compared to the percentage pre-on-call (P = 0.001). The post-on-call total POMS scores of groups 1 and 2 were significantly worse than their pre-on-call scores (P = 0.001 and 0.038, respectively), while there was no significant difference between the pre- and post-on-call POMS scores of group 3 (P = 0.165). Conclusion: Acute sleep loss due to working long on-call shifts significantly decreases daytime alertness and negatively affects the mood state of junior physicians.