Heart failure with preserved ejection fraction (HFpEF) is characterized by a progressive decline in diastolic cardiac function with a normal ejection fraction (EF). Furthermore, sympathoexcitation in HFpEF has been linked to cardiac arrhythmias. We recently described that activation of the central chemoreflex pathway (CC) induced cardiac autonomic imbalance. Therefore, we aimed to determine whether the retrotrapezoid (RTN) nuclei, a main chemoreceptive area, and the rostral ventrolateral medulla (RVLM), a major region involved in the regulation of sympathetic outflow, displayed chronic neuronal activation in the setting of HFpEF. Furthermore, we explored if both inflammation and angiotensin II signalling pathways could be associated with the changes in neuronal activity in the RTN and RVLM from rats with HFpEF. Male Sprague-Dawley rats were anesthetized (isoflurane 2% in O2) and subjected to aorto-caval shunt to induce HFpEF. Ventilatory response to acute hypercapnia (FiCO2 7%) was assessed to determine CC sensitivity. Finally, rats were anesthetized (α-chloralose 40 mg/kg and urethane 800 mg/kg i.p.) and cardiac function was determined by pressure-volume loops. Neuronal activation was assessed in RTN and RVLM micropunches by measuring FosB expression by immunoblot. Compared to Sham rats, HFpEF rats display (HFpEF vs. Sham): normal EF (51±3 vs. 50±7 %), cardiac hypertrophy (heart to body weight ratio, 6.1±0.3 vs. 4.0±0.5 mg/g; P<.05), increased arrhythmia incidence (196±84 vs. 19±7 events/h; P<.05) and increased end diastolic pressure-volume relationship (β, 0.0076±0.001 vs. 0.0039±0.001 1/ul; P<.05). In addition, we found that HFpEF rats display enhanced CC sensitivity compared to Sham animals (165.3±9.1 vs. 127.3±10.3 ml/min/100g, HFpEF vs. sham, respectively; P<.05). Despite the significant increase in CC gain, HFpEF rats showed no changes in normoxic minute ventilation compared to Sham. RVLM FosB expression was increased by ~2.5 fold in HFpEF compared to sham rats. On the contrary, no change in FosB in the RTN was found between groups. In order to determine the plausible mechanisms that could mediate the RVLM hyper activation we screened for changes in angiotensin II type 1 receptor (AT1R) and for p65 subunit of the NF-kB pathway. We found an augmented expression of AT1R in the RVLM from HFpEF rats compared to sham rats (276±48% vs. 100±20%, HFpEF vs. sham, respectively; P<.05). NF-kB p65 expression was also increased in the RVLM of HFpEF. Our results show that autonomic imbalance in HFpEF is associated with chronic neuronal activation of the RVLM but not with the activation of the RTN. Furthermore, AT1R and p65 are highly expressed in the RVLM of HFpEF rats. Our results suggest that central angiotensin II and inflammation may partially play a role in RVLM activation and sympathoexcitation in HFpEF.