The mitral early to late diastolic flow velocity ratio (E/A ratio) is age-dependent. It has been considered that its age dependency reflects the age-related lengthening of left ventricular (LV) relaxation; however, the change in E/A ratio is far larger than that expected from those in LV relaxation. We hypothesized that an age-related reduction of the parasympathetic activity increases left atrial (LA) contractility, and that this accounts for the age-related change in E/A ratio. (1) Exercise stress test was performed in 61 normal subjects (age range, 8-80 years, mean, 40 years) to assess heart rate (HR) recovery because slowed HR recovery indicates lowered parasympathetic activity. There were good interrelations among age, E/A ratio, and HR recovery. Among those aged ≤30 years, the age no longer correlated with E/A ratio or HR recovery, but there was a significant correlation between HR recovery and E/A ratio (r = 0.44, p < 0.05). (2) Pulsed Doppler and two-dimensional speckle tracking echocardiography (2DSTE) were performed before and after administration of parasympathetic blockade (atropine) in ten young healthy subjects. LA booster pump function was assessed with LA emptying index calculated by 2DSTE. LA emptying index was calculated from ([LA volume before the atrial contraction - minimal LA volume]/LA volume before the atrial contraction) × 100. Atropine increased mitral A velocity (p < 0.001) and LA emptying index (p < 0.05) along with a decrease in E/A ratio (p < 0.001). Parasympathetic withdrawal enhances LA contraction and increases mitral A velocity, which likely cause a reciprocal decrease in mitral E velocity and E/A ratio. Thus, parasympathetic deactivation with aging should be closely involved in the age-related change in mitral E/A ratio.