Clinically, healthy hearts have been associated with a high ventricular heart rate variability (HRV) while diseased hearts have been known to exhibit low ventricular HRV. Hence, low HRV is suggested to be a marker of cardiac ventricular arrhythmias. Over the past few years, there has been considerable amount of interest in incorporating HRV in pacing to emulate healthy heart conditions and re-stabilize the electrical activity in diseased hearts. Recently, we used single cell numerical simulations to demonstrate that HRV incorporated into periodic pacing promotes alternans formation and thus, can be pro-arrhythmic. Here, we performed high-resolution optical mapping experiments on Langendorff perfused, healthy whole mice hearts to validate our numerical findings. Our results indeed demonstrate that HRV promoted the onset of cardiac alternans, which is believed to be a precursor of fatal cardiac rhythms. Hence, our present study suggests that incorporating HRV into periodic pacing while addressing several clinical needs may not be safe. There is a pressing need to better understand paced cardiac dynamics and develop anti-arrhythmic pacing techniques that would prevent cardiac arrhythmias.