Abstract Background Vagal responses are frequently observed during pulmonary vein isolation (PVI). Radiofrequency ablation of the anterior right ganglionated plexus (ARGP) before PVI has been demonstrated to effectively prevent VR occurrence. Pulsed-field ablation (PFA) has emerged as an alternative energy source for PVI. Despite PFA being selective for the myocardium, intraprocedural VRs and reversible effects on ganglionated plexi (GP) have been documented during PVI. Purpose We aimed to investigate whether initiating PVI from the right superior pulmonary vein (RSPV) before any other pulmonary veins (PVs) could prevent VR occurrence via concomitant transient ARGP modulation. Methods 60 consecutive paroxysmal atrial fibrillation patients undergoing PVI with the pentaspline PFA ablation catheter were prospectively included. In the first 30 patients, PVI was started from the left superior pulmonary vein (LSPV-first group). In the last 30 patients, the RSPV was targeted first, followed by the left PVs, and finally the right inferior PV (RSPV-first group). VR was defined as sinus bradycardia (<40 beats/min), asystole, or atrioventricular block (AVB). Heart rate (HR) was evaluated at baseline, during PVI, and post-ablation to assess GP modulation. Results Baseline characteristics were similar between the two study groups. VR during PVI occurred in 22 (73%) patients in the LSPV-first group and 3 (10%) in the RSPV-first group (Figure1, p<0.001). A higher rate of VRs was observed in patients in the LSPV-first group compared to patients in the RSPV-first group during ablation of the LSPV and LIPV (73% vs. 7%, p<0.001 and 27% vs. 0%, p=0.005, respectively), while no differences were found during ablation of the right PVs. Temporary pacing was employed in 10 (33%) patients in the LSPV-first group and 3 (10%) in the RSPV-first group (p=0.028). RSPV isolation induced similar acute HR increase in the two groups (13±9 bpm vs 13±10 bpm, p=0.9). However, no significant HR variation was documented in both groups at the end of the procedure compared to baseline (all p>0.05). Conclusion PVI with PFA is frequently associated with intraprocedural VRs when ablation is initiated from the LSPV. However, an RSPV-first approach determined transient HR increase and reduced VR occurrence.Figure 1