Maintaining vigilance over long periods of time is especially critical in performing fundamental everyday activities and highly responsible professional tasks (e.g., driving, performing surgery or piloting). Here, we investigated the role of aerobic fitness as a crucial factor related to the vigilance capacity. To this end, two groups of young adult participants (high-fit and low-fit) were compared in terms of reaction time (RT) performance and event-related heart rate responses in a 60' version of the psychomotor vigilance task. The results showed shorter RTs in high-fit participants, but only during the first 24' of the task. Crucially, this period of improved performance was accompanied by a decelerative cardiac response pattern present only in the high-fit group that also disappeared after the first 24'. In conclusion, high aerobic fitness was related to a pattern of transient autonomic responses suggestive of an attentive preparatory state that coincided with improved behavioural performance, and that was sustained for 24'. Our findings highlight the importance of considering the role of the autonomic nervous system reactivity in the relationship between fitness and cognition in general, and sustained attention in particular.