The purpose of this study was to evaluate if the time of the day (8.00 a.m. vs 8.00 p.m.) and chronotype could influence autonomic cardiac control in soccer players in relation to an acute session of high-intensity interval training. The morningness-eveningness questionnaire was administered to recruit Morning-type and Evening-type collegiate male soccer players. Therefore, 24 players (12 Morning-types and 12 Evening-types) were randomly assigned, to either morning (n = 12; age 23 ± 3 years; height 1.75 ± 0.07 m; body mass 73 ± 10 kg; weekly training volume 8 2 hours), or evening (n = 12; age 21 ± 3 years; height 1.76 ± 0.05 m; body mass 75 ± 11 kg; weekly training volume 8 ± 3 hours) training. Heart Rate Variability vagal and sympatho/vagal indices were calculated in time, frequency and complexity domains at rest, before, after 12 and 24 hours of high-intensity interval training. Before evening training session, a higher resting heart rate was observed which was determined by a marked parasympathetic withdrawal with a sympathetic predominance. Moreover, Evening-type subjects during morning training session, present a significant higher heart rate that corresponded to significant higher vagal indices with a significant lower parasympathetic tone that returned to the rest values after 24 hours of the cessation of high-intensity interval training exercise. On the contrary, Morning-type subjects did not reveal any significant differences with Evening-Type subjects during evening high-intensity interval training session. Stress response of high-intensity interval training is influenced by both the time of the day and by the chronotype. Understanding the Heart Rate Variability response to high-intensity interval training can be an additional important procedure for evaluating of cardiovascular recovery in soccer players. Moreover, these results suggest that an athlete’s chronotype should be taken into account when scheduling a high-intensity interval training exercise.