A reliable non-invasive method to assess autonomic nervous system activity involves the evaluation of the time course of heart rate variability (HRV). HRV may vary in accordance with the degree and duration of training, and the circadian fluctuation of this variable is crucial for human health since the heart adapts to the needs of different activity levels during sleep phases or in the daytime. In the present review, time-of-day and chronotype effect on HRV in response to acute sessions of physical activity are discussed. Results are sparse and controversial; however, it seems that evening-type subjects have a higher perturbation of the autonomic nervous system (ANS), with slowed vagal reactivation and higher heart rate values in response to morning exercise than morning types. Conversely, both chronotype categories showed similar ANS activity during evening physical tasks, suggesting that this time of day seems to perturb the HRV circadian rhythm to a lesser extent. The control for chronotype and time-of-day effect represents a key strategy for individual training schedules, and, in perspective, for primary injury prevention.