In 106 community-dwelling middle-aged non-smokers we examined the time-course and the acute effects of fine particles (PM₂.₅) on heart rate variability (HRV), which measures cardiac autonomic modulation (CAM). Twenty-four hours beat-to-beat ECG data were visually examined. Artifacts and arrhythmic beats were removed. Normal beat-to-beat RR data were used to calculate HRV indices. Personal PM₂.₅ nephelometry was used to estimate 24-h individual-level real-time PM₂.₅ exposures. We use linear mixed-effects models to assess autocorrelation- and other major confounder-adjusted regression coefficients between 1-6 h moving averages of PM₂.₅ and HRV indices. The increases in preceding 1-6 h moving averages of PM₂.₅ was significantly associated with lower HF, LF, and SDNN, with the largest effect size at 4-6 h moving averages and smallest effects size at 1 h moving average. For example, a 10 μg/m³ increase in 1 and 6-h moving averages was associated with 0.027 and 0.068 ms² decrease in log-HF, respectively, and with 0.024 and 0.071 ms² decrease in log-LF, respectively, and with 0.81 and 1.75 ms decrease in SDNN, respectively (all P-values <0.05). PM₂.₅ exposures are associated with immediate impairment of CAM. With a time-course of within 6 h after elevated PM₂.₅ exposure, with the largest effects around 4-6 h.