Aims Cardiac resynchronization therapy with biventricular pacing has proved beneficial in symptomatic heart failure patients, yet the effects in patients with structurally normal hearts remain unknown. We hypothesized that, in an acute swine model with normal anatomy and function, single-site right ventricular (RV) pacing would better preserve haemodynamic function and electrical activation compared to biventricular pacing. Methods Endocardial single-site pacing was performed in anesthetized swine (n = 7) from the RV septum and RV apex. Biventricular pacing was performed using an epicardial left ventricular (LV) lead and a RV lead. High-resolution, non-contact mapping was employed to record LV activation sequences simultaneously with haemodynamic data after 5 min of consistent capture. Results All pacing interventions significantly prolonged QRS and total endocardial activation durations (P < 0.05) compared to intrinsic activation. Biventricular pacing with the RV apex lead significantly impaired LV systolic mechanics (dP/dt(max), max LV pressure; P < 0.05), and reduced LV relaxation to the greatest extent (dP/dt(min), P = ns). Right ventricular septal pacing conserved function better than other pacing interventions (P = ns) and elicited an intrinsic electrical activation sequence. Conclusion In intact, synchronous hearts, acute biventricular pacing resulted in systolic dysfunction and abnormal LV electrical activation.