Most animals look at each other to signal threat or interest. In humans, this social interaction is usually punctuated with brief periods of mutual eye contact. Deviations from this pattern of gazing behaviour generally make us feel uncomfortable and are a defining characteristic of clinical conditions such as autism or schizophrenia, yet it is unclear what constitutes normal eye contact. Here, we measured, across a wide range of ages, cultures and personality types, the period of direct gaze that feels comfortable and examined whether autonomic factors linked to arousal were indicative of people's preferred amount of eye contact. Surprisingly, we find that preferred period of gaze duration is not dependent on fundamental characteristics such as gender, personality traits or attractiveness. However, we do find that subtle pupillary changes, indicative of physiological arousal, correlate with the amount of eye contact people find comfortable. Specifically, people preferring longer durations of eye contact display faster increases in pupil size when viewing another person than those preferring shorter durations. These results reveal that a person's preferred duration of eye contact is signalled by physiological indices (pupil dilation) beyond volitional control that may play a modulatory role in gaze behaviour.