Diastolic dysfunction is important in the pathophysiology of heart failure with preserved ejection fraction (HFpEF). Sympathetic nervous hyperactivity may contribute to the development of diastolic dysfunction. The aim of this study was to determine the relationship between myocardial sympathetic innervation quantified by 11C-hydroxyephedrine PET and diastolic dysfunction in HFpEF patients. Methods: Forty-one HFpEF patients having an echocardiographic left ventricular ejection fraction of 40% or greater and 12 age-matched volunteers without heart failure underwent the echocardiographic examination and 11C-hydroxyephedrine PET. Diastolic dysfunction was classified into grades 0-3 by Doppler echocardiography. Myocardial sympathetic innervation was quantified using the 11C-hydroxyephedrine retention index (RI). The coefficient of variation of 17-segment RIs was derived as a measure of heterogeneity in myocardial 11C-hydroxyephedrine uptake. Results: Grade 2-3 diastolic dysfunction (DD2-3) was found in 19 HFpEF patients (46%). They had a significantly lower global RI (0.075 ± 0.018 min-1) than volunteers (0.123 ± 0.028 min-1, P < 0.001) and HFpEF patients with grade 0-1 diastolic dysfunction (DD0-1) (0.092 ± 0.024 min-1, P = 0.046). HFpEF patients with DD2-3 had the largest coefficient of variation of 17-segment RIs of the 3 groups (18.4% ± 7.7% vs. 14.1% ± 4.7% in HFpEF patients with DD0-1, P = 0.042 for post hoc tests). In multivariate logistic regression analysis, a lower global RI (odds ratio, 0.66 per 0.01 min-1; 95% confidence interval, 0.38-0.99; P = 0.044) was independently associated with the presence of DD2-3 in HFpEF patients. Conclusion: Myocardial sympathetic innervation was impaired in HFpEF patients and was associated with the presence of advanced diastolic dysfunction in HFpEF.