Short sleep (SS) disrupts the normal functioning of the autonomic nervous system (ANS). Heart rate variability (HRV) is a reproducible measure of ANS function. Reduction in HRV represents a disruption of the ANS. High-intensity interval exercise (HIIE) reduces HRV. The immediate HRV response and its short-term recovery after HIIE is unexplored. SS prior to HIIE may disrupt further the ANS and reduce HRV. PURPOSE: To characterize the influence of SS on HRV after a single bout of HIIE in the hours post-exercise (PE). METHODS: Fifteen physically active males (age 31.1±5.3 yr) with good sleep quality as determined by the Pittsburgh Sleep Quality Index (PSQI) participated in this study. Participants completed a non-exercise control trial after 9 to 9.5 hrs of reference sleep (RS), a HIIE treadmill running (90% and 40% of VO₂reserve in 3:2 min ratio) to expend 500 kcals after reference sleep (RSX) and after 3 to 3.5 hrs of SS (SSX) in a randomized crossover design. After being in a supine position for 10 minutes in a quiet and temperature-controlled environment heart rate (HR) was recorded for 5 minutes the night before, the morning of the next day, 1-, 2-, 4- and 6-hours PE using an elastic electrode belt (Polar Wearlink^®). Sleep was performed at their own residence. Supine resting HRV indices (frequency domain: LF/HF and VLF) obtained from HR were measured and processed by CardioMood^®. Data were analyzed using a 3 (condition) by 6 (time) repeated measures ANOVA. Examined variables violated normality and thus were transformed into natural logarithm (ln). Bonferroni was used for post hoc comparisons. Significance was set at p < 0.05. All analyses were performed using SPSS^©. RESULTS: For ln VLF there was a main effect for condition by time interaction (F_10,140=3.60, p=.000, η²=.204), but not for ln LF/HF condition by time (F_10,140=1.84, p=.060, η²=.116). ln VLF was both decreased in RSX at 1hr PE (6.3+.25, p=.001) and 2hr PE (7.1+.20, p=.007) and in SSX 1hr PE (6.1+.22, p=.000) and 2hr PE (7.4+.26, p=.000) compared to RS. During post hocanalysis frequency domain ln LF/HF was increased in SSX at Day 2-baseline (.29+.17, p=.040) and at 1hr PE (1.1+.19, p=.041). CONCLUSION: HIIE with 3:2 min ratio decreases HRV for up to 2 hours PE and takes more than 6 hours to return to baseline levels. These responses are not modified by a single night of short sleep.