Acute ambient ozone (O(3)) exposure is associated with the increased mortality and morbidity of cardiovascular diseases. The dysfunction of cardiac autonomic nervous system (ANS), indicated by the disturbed heart rate variability (HRV), may be the most important underlying mechanism. Previous studies reported the heterogeneous associations between O(3) within several hours' exposure and HRV on general elderly subjects, in which poor surrogate of exposure evaluation and different health status of the subjects may be responsible for the heterogeneous associations. No studies were found focusing on the O(3)-mediated HRV effects within several minutes' exposure on healthy older subjects until recently. We measured the real-time 5-min ambient O(3) concentration and HRV frequency indices in 20 healthy elderly subjects in two surveys, with the 1st and 2nd survey in summer and winter, respectively. Mixed-linear model was used to evaluate the associations between the ambient 5-min average O(3) and concurrent 5-min HRV frequency indices measured during the outdoor period. After adjusting the co-pollutants (ambient PM(2.5) and nitrogen oxides concentrations) and subject characteristics, high frequency (HF) changed -4.87% (95% CI -8.62 to -0.97%) per 10 ppb increment of O(3), whereas decreased low frequency (LF) and increased LFHFR were found to be marginally associated with the elevated O(3) (P values were 0.092 and 0.069). We concluded that the ambient O(3) exert transient decrease effects on HRV, which may induce acute cardiac events.