Biological circadian rhythms have an important influence on ventricular function. The mechanistic basis for the morning prevalence of cardiac arrhythmias and sudden cardiac death is unclear, but may reflect a combination of increased sympathetic activity and diurnal variation of genes important for cardiac excitability. In the current study, we investigated whether there is any diurnal variation in single cell electrical restitution under basal conditions and with β-adrenergic receptor stimulation. Electrical restitution is the relationship between action potential duration (APD) and diastolic interval (DI) and describes APD adaptations at higher heart rates. In the intact heart, a steep slope of the restitution curve is associated with ventricular fibrillation. Left ventricular myocytes were isolated from adult male guinea pigs (500-700 g) at two opposing time points, corresponding to the animals in resting or active periods. Action potentials were recorded at 35-37 °C using the amphotericin-perforated patch-clamp technique. Electrical restitution data were obtained using pacing cycle lengths (PCL) that were shortened until the effective refractory period (ERP) was reached. APD was allowed to stabilize at each PCL and the mean APD at 90% repolarisation (APD90) and DI determined from 20 events. The dynamic pacing protocol was repeated in the presence of isoprenaline (Iso, 10 nM) and Iso plus the non-selective nitric oxide synthase (NOS) inhibitor N-nitro-L-arginine (NNA, 500 µM). Restitution curves were fitted individually with exponential functions and mean (± SEM) values for time constants (τ), maximum APD90 and slope at shortest DI calculated. No significant difference (p > 0.05) in maximum APD90 was observed under basal conditions in active (n = 20) versus resting period (n = 24) myocytes. However, there were differences in the shapes of the electrical restitution curves in there basal conditions. τ-values were significantly (p<0.01) shorter (191.8 ± 29.9 ms n=18, and 304.7 ± 29.9 ms n=20) and maximum slopes steeper (1.0 ± 0.08 n=17, and 0.7± 0.08 n= 19) for active versus resting period myocytes respectively. ERPs were also significantly (P<0.05) shorter in active (147 ±0.009 ms, n =16) than resting period (177 ± 0.009 ms, n =11) myocytes. Although resting period myocytes responded more to Iso, maximum slopes were not as steep and ERPs not as short as in active period myocytes. Arrhythmic events (alternans and delayed after-depolarizations) were detected in 12 active and 3 resting period myocytes. Active period cells responded significantly more to NNA. In conclusion, diurnal variations were observed in single cell electrical restitution, responses to β-adrenergic receptor stimulation and NOS activity. Our results indicate active period myocytes are more susceptible to arrhythmias than resting period ones