BACKGROUND: Ultra-short-term heart rate variability (HRV) analysis (< 5鈥痬in) has been extensively growing in the field of exercise performance for autonomic assessment. However, the validation of ultra-short-term HRV was unclear in the recovery period of exercise. This study aimed to elucidate the agreement between ultra-short-term HRV (0-30鈥痵, 0-1鈥痬in, 0-2鈥痬in, 0-3鈥痬in, 0-4鈥痬in) and standard short-term HRV (5鈥痬in) and to explore the optimal recording duration under rest and post-exercise conditions. METHODS: 69 participants were recruited to perform physical exercise on a treadmill with an intensity of 6鈥痥m/h, 9鈥痥m/h and 12鈥痥m/h, independently. The standard deviation of RR-interval (SDNN) and root mean square of successive differences of RR-intervals (RMSSD) were calculated by using ultra-short periods and standard period at rest condition (Pre-E) and three post-exercise trials, i.e., Post-E1, Post-E2 and Post-E3, respectively. One-way ANOVA with repeated-measures and Cohen\'s d statistics were conducted, and Bland-Altman analysis and interclass correlation coefficients (ICC) were used to assess the levels of agreement. RESULTS: For SDNN and RMSSD, the results of agreement analysis at rest condition were different from those at post-exercise. At Pre-E, SDNN and RMSSD were reliable for ultra-short-term HRV analysis at all ultra-short periods, i.e., 0-30鈥痵, 0-1鈥痬in, 0-2鈥痬in, 0-3鈥痬in and 0-4鈥痬in, with most ICCs greater than 0.9 and Cohen\'s d showing trivial differences (Cohen\'s d鈥?鈥?.024-0.117). However, at post-exercise, SDNN(0-30s), SDNN(0-1min), RMSSD(0-30s) and RMSSD(0-1min) showed significant differences with SDNN(5min) and RMSSD(5min), respectively (p鈥?鈥?.01), and the ICCs was not perfect (< 0.9). HRV analysis with time duration longer than 2 min showed nearly perfect reliability in all post-exercise trials, with trivial differences (Cohen\'s d鈥?鈥?0.003-0.110) and perfect ICCs (ICCs鈥?鈥?.916-0.998). Furthermore, the limits of the agreement became tighter as the period duration increased in Bland-Altman plots. CONCLUSIONS: This study demonstrated that ultra-short-term HRV analysis was a good surrogate of standard HRV time-domain measures to reflect the autonomic regulation at rest and post-exercise. Specifically, ultra-short-term HRV(0-30s) or HRV(0-1min) was recommended at rest condition, whereas longer than 2 min recording period was reliable to obtain SDNN and RMSSD for the accuracy of HRV analysis.