Stress often co-occurs with inadequate sleep duration, and both are believed to impact mood and emotion. It is not yet known whether inadequate sleep simply increases the intensity of subsequent stress responses or interacts with stressors in more complicated ways. To address this issue, we investigated the effects of one night of total sleep deprivation on subjective stress and mood in response to low-stress and high-stress cognitive testing conditions in healthy adult volunteers in two separate experiments (total N = 53). Sleep was manipulated in a controlled, laboratory setting and stressor intensity was manipulated by changing difficulty of cognitive tasks, time pressure, and feedback about performance. Sleep-deprived participants reported greater subjective stress, anxiety, and anger than rested controls following exposure to the low-stressor condition, but not in response to the high-stressor condition, which elevated negative mood and stress about equally for both sleep conditions. These results suggest that sleep deprivation lowers the psychological threshold for the perception of stress from cognitive demands but does not selectively increase the magnitude of negative affect in response to high-stress performance demands.