Introduction: Emotion regulation and empathy are affected by sleep deprivation. Whether these effects are caused by changes in neural activity in regions regulating the functions is unknown. When well-rested, cortical activity regulates emotional reactions and empathy by decreasing activity in the amygdalae. Neural activity can be measured with functional magnetic resonance imaging (fMRI). Aim: The aim of this study was to investigate the effect of one night of sleep deprivation on empathy for pain and emotion regulation through cognitive reappraisal, that is, intentional downregulation. We hypothesized that sleep deprivation would lead to decreased cortical activity and increased activity in amygdalae. Material and Methods: 35 healthy, young participants underwent fMRI after one night of sleep deprivation and one night of habitual sleep. Behaviorally, empathy was assessed by comparing self-reported unpleasantness between painful images and non-painful images, and reappraisal was investigated by comparing unpleasantness between instructions to downregulate and maintain. fMRI was performed to measure neural activity during the task. Results: Participants reported less unpleasantness when viewing painful images when sleep-deprived (p < 0.001). Sleep deprivation caused no decrease in ability to reappraise, nor did sleep deprivation cause decrease in cortical fMRI-signals. Activity in the amygdalae was non-significantly increased in sleep-deprived condition (p = 0.13). Conclusions: As hypothesized, the empathic response to pictures of pain was decreased during sleep deprivation. However, no decrease in ability to reappraise was found. Sleep deprivation did not cause changes in neural activity that would support our hypothesis of decreased inhibitory cortical activity.