<jats:title>Abstract</jats:title><jats:sec><jats:title>Aims</jats:title><jats:p>In heart failure with preserved ejection fraction (HFpEF), the reduction of nitric oxide (NO)‐bioavailability and consequently endothelial dysfunction leads to LV stiffness and diastolic dysfunction of the heart. Besides shear stress, high‐density lipoprotein (HDL) stimulates endothelial cells to increased production of NO via phosphorylation of endothelial nitric oxide synthase (eNOS). For patients with heart failure with reduced ejection fraction, earlier studies demonstrated a positive impact of exercise training (ET) on HDL‐mediated eNOS activation. The study aims to investigate the influence of ET on HDL‐mediated phosphorylation of eNOS in HFpEF patients.</jats:p></jats:sec><jats:sec><jats:title>Methods and results</jats:title><jats:p>The present study is a substudy of the OptimEx‐Clin trial. The patients were randomized to three groups: (i) HIIT (high‐intensity interval training; (ii) MCT (moderate‐intensity continuous training); and (iii) CG (control group). Supervised training at study centres was offered for the first 3 months. From months 4–12, training sessions were continued at home with the same exercise protocol as performed during the in‐hospital phase. Blood was collected at baseline, after 3, and 12 months, and HDL was isolated by ultracentrifugation. Human aortic endothelial cells were incubated with isolated HDL, and HDL‐induced eNOS phosphorylation at Ser<jats:sup>1177</jats:sup> and Thr<jats:sup>495</jats:sup> was assessed. Subsequently, the antioxidative function of HDL was evaluated by measuring the activity of HDL‐associated paraoxonase‐1 (Pon1) and the concentration of thiobarbituric acid‐reactive substances (TBARS). After 3 months of supervised ET, HIIT resulted in increased HDL‐mediated eNOS‐Ser<jats:sup>1177</jats:sup> phosphorylation. This effect diminished after 12 months of ET. No effect of HIIT was observed on HDL‐mediated eNOS‐Thr<jats:sup>495</jats:sup> phosphorylation. MCT had no effect on HDL‐mediated eNOS phosphorylation at Ser<jats:sup>1177</jats:sup> and Thr<jats:sup>495</jats:sup>. HIIT also increased Pon1 activity after 12 months of ET and reduced the concentration of TBARS in the serum after 3 and 12 months of ET. A negative correlation was observed between TBARS concentration and HDL‐associated Pon1 activity in the HIIT group (<jats:italic>r</jats:italic> = −0.61, <jats:italic>P</jats:italic> < 0.05), and a trend was evident for the correlation between the change in HDL‐mediated eNOS‐Ser<jats:sup>1177</jats:sup> phosphorylation and the change in peak V̇O<jats:sub>2</jats:sub> after 3 months in the HIIT group (<jats:italic>r</jats:italic> = 0.635, <jats:italic>P</jats:italic> = 0.07).</jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p>The present study documented that HIIT but not MCT exerts beneficial effects on HDL‐mediated eNOS phosphorylation and HDL‐associated Pon1 activity in HFpEF patients. These beneficial effects of HIIT were reduced as soon as the patients switched to home‐based ET.</jats:p></jats:sec>