Very short-term heart rate variability (HRV) is thought to reflect dynamic changes in autonomic nervous activity, which is helpful in understanding the role of autonomic nervous function (ANF) in the mechanisms underlying apnea-induced cardiac arrhythmias. The goal of this study was to investigate the effect of repetitive end-inspiration breath holding on very short-term HRV. A total of 32 young healthy participants took part in the experiments. Three trials were performed, each involving seven repetitive end-inspiration breath holding and a 30 s recovery period between breath holding. Durations of breath holding in the three trials were 1:2:3. The study first evaluated the effect of analyzed data lengths on the stability of HRV indices and determined three HRV indices suitable for very short-term analysis. The results showed that in most cases, during breath holding, the square root of the mean squared differences of successive normal RR intervals (rMSSD) was significantly lower, but normalized units of the power in the low frequency band ranging from 0.04 to 0.15 Hz (nLF) and LF/high frequency (HF) were significantly higher than those during corresponding durations under the normal breathing conditions. On the contrary, during recovery after breath holding, rMSSD was significantly higher but nLF and LF/HF were lower than normal. Moreover, the durations of breath holding had no significant influence on the variations of LF/HF. In addition, as participants repeated the breath holding, HRV indices varied non-linearly. HRV changes may indicate sympathetic activation during breath holding and parasympathetic activation during recovery after breath holding. In conjunction with the existing physiological interpretation based on changes in heart rate, the results may imply that breath holding leads to both cardiac sympathetic and parasympathetic activation simultaneously, which may be a possible pathogenic factor of apnea-induced arrhythmias.