Heart failure is characterized by sympathetic activation and parasympathetic withdrawal leading to an abnormal autonomic modulation. Beta-blockers (BB) inhibit overstimulation of the sympathetic system and are indicated in heart failure patients with reduced ejection fraction. However, the effect of beta-blocker therapy on heart failure with preserved ejection fraction (HFpEF) is unclear. This study investigated the effect of BB therapy on heart rate variability (HRV) features as a measure of risk of an abnormal cardiac event. ECGs of seventy-three patients with HFpEF > 55% were recruited. Fifty-six patients in the BB group and 17 patients in the without BB group. HRV analysis was performed for recordings between 6-10 am and 6-10 pm, which are times associated with increased risk of cardiac events. The result shows that RMSSD (p=0.011), HF power (p=0.012), and VLF power (p=0.047) were significantly higher during the 6 – 10 am interval. Sample entropy (p=0.016), and the novel fragmentation measures PIP (p=0.015), IALS (p=0.015) and PSS (p=0.008) were significantly higher between 6 – 10 pm. Beta-blocker therapy increases HRV measures in the HFpEF group depending on the feature investigated indicating an overall decreased risk of a cardiac event and a possibly beneficial effect of beta-blockers, especially during the morning hours that is characterized by a sympathetic surge.