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Background: It remains unclear which size of particles has the strongest effects on heart rate variability (HRV). Objective: To explore the association between HRV parameters and daily variations of size-fractionated particle number concentrations (PNCs). Methods: We conducted a longitudinal repeated-measure study among 78 participants with a 24-h continuous ambulatory Holter electrocardiographic recorder in Shanghai, China, from January 2015 to June 2019. Linear mixed-effects models were employed to evaluate the changes of HRV parameters associated with PNCs of 7 size ranges from 0.01 to 10 μm after controlling for environmental and individual confounders. Results: On the concurrent day, decreased HRV parameters were associated with increased PNCs of 0.01–0.3 μm, and smaller particles showed greater effects. For an interquartile range increase in ultrafine particles (UFP, those < 0.1 μm, 2453 particles/cm3), the declines in very-low-frequency power, low-frequency power, high-frequency power, standard deviation of normal R-R intervals, root mean square of the successive differences between R-R intervals and percentage of adjacent normal R-R intervals with a difference ≥ 50 ms were 5.06% [95% confidence interval (CI): 2.09%, 7.94%], 7.65% (95%CI: 2.73%, 12.32%), 9.49% (95%CI: 4.64%, 14.09%), 5.10% (95%CI: 2.21%, 7.91%), 8.09% (95%CI: 4.39%, 11.65%) and 24.98% (95%CI: 14.70%, 34.02%), respectively. These results were robust to the adjustment of criteria air pollutants, temperature at different lags, and the status of heart medication. Conclusions: Particles less than 0.3 μm (especially UFP) may dominate the acute effects of particulate air pollution on cardiac autonomic dysfunction.