In mobile healthcare, heart rate variability (HRV) is increasingly being used in dynamic patient states. In this situation, shortening of the measurement time is required. This study aimed to validate ultra-short-term HRV in non-static conditions. We conducted electrocardiogram (ECG) measurements at rest, during exercise, and in the post-exercise recovery period in 30 subjects and analyzed ultra-short-term HRV in time and frequency domains by ECG in 10, 30, 60, 120, 180, and 240-s intervals, and compared the values to the 5-min HRV. For statistical analysis, null hypothesis testing, Cohen’s d statistics, Pearson’s correlation coefficient, and Bland-Altman analysis were used, with a statistical significance level of P < 0.05. The feasibility of ultra-short-term HRV and the minimum time required for analysis showed differences in each condition and for each analysis method. If the strict criteria satisfying all the statistical methods were followed, the ultra-short-term HRV could be derived from a from 30 to 240-s length of ECG. However, at least 120 s was required in the post-exercise recovery or exercise conditions, and even ultra-short-term HRV was not measurable in some variables. In contrast, according to the lenient criteria needed to satisfy only one of the statistical criteria, the minimum time required for ultra-short-term HRV analysis was 10–60 s in the resting condition, 10–180 s in the exercise condition, and 10–120 s in the post-exercise recovery condition. In conclusion, the results of this study showed that a longer measurement time was required for ultra-short-term HRV analysis in dynamic conditions. This suggests that the existing ultra-short-term HRV research results derived from the static condition cannot applied to the non-static conditions of daily life and that a criterion specific to the non-static conditions are necessary.