Well-powered studies on the physiological concomitants underlying affect and its regulation during emerging adulthood are warranted to provide novel insight into mental health. The association between autonomic nervous system activity and emotional states occurring under natural conditions in daily life was investigated in individuals (N = 549, age 14–21, females 45.6%) without any lifetime mental disorder from an epidemiological cohort study in Germany. Using ecological momentary assessment, mood and optimism/pessimism were assessed over 4 days simultaneously with continuous heart rate monitoring. Lower vagal activity was found in mood states accompanied by high arousal (wakefulness, mania) and greater vagal activity in mood states with low arousal (calmness, pessimism). Findings illustrate important associations between autonomic nervous system activity and mood in youth under ecologically valid conditions. Vagal activity presents a prominent pathway by which mood may influence physiological function or vice versa. In contrast to commonly performed laboratory assessments, the ambulatory assessment in participants' daily life allows an application of the results to the field.