Many pathological cardiovascular events show morning prevalence, possibly reflecting diurnal changes in cardiac haemodynamics, metabolism and sympathetic activity. Whilst around 10% of rat cardiac gene expression displays diurnal variations, little is known of whether this impacts on cardiac excitation-contraction (E-C) coupling. We have, therefore, set out to determine whether cardiac E-C coupling shows diurnal variation and if this influences the arrhythmia threshold of ventricular tissue to sympathetic stimulation. Single ventricular myocytes were isolated from hearts excised at two opposing timepoints, ZT3 and ZT15, where ZT0 refers to “lights on”. [Ca²⁺]_i was measured using Fura-2, cell shortening with a cell-edge detection system and L-type Ca²⁺ current density using the whole cell, patch clamp technique. The data show that basal percent cell shortening and systolic [Ca²⁺]_i was significantly higher in ZT3 than ZT15 myocytes; with a % cell shortening of 12.4±0.3 in ZT3 myocytes (n=209) vs 11.0±0.2 (n=216) in ZT15 myocytes (S.E.M; P<0.05), and a peak systolic [Ca²⁺]_i of 422±12nM (n=166) in ZT3 vs 341±9nM (n=176) in ZT15 myocytes (S.E.M; students t-test, P<0.01). The SR Ca²⁺ content revealed by the application of 20mM caffeine was significantly higher in ZT3 myocytes, with a peak Ca²⁺ of 672.8 ± 20.5nM (n=71) vs 550.9±12.9 (n=97) in ZT15 myocytes (S.E.M; students t-test, P<0.001). We looked at β-adrenergic stimulation with isoproterenol (ISO) to simulate sympathetic activity. We found no significant difference in EC₅₀ of ISO-stimulation of systolic Ca²⁺, but a significant reduction in the steady-state response at concentrations >3nM in ZT15 myocytes, with a maximum systolic [Ca²⁺]_i, recorded in 100nM ISO, of 2,330 ± 402nM (n=33) in ZT3 myocytes vs. 1,384 ± 109nM (n=36) in ZT15 myocytes (S.E.M; students t-test, P<0.01). A similar diurnal variation was shown in the response of the L-type Ca²⁺ current to ISO, with a maximal percentage increase in current density of 67.0%±8.5 (n=15) in ZT3 myocytes compared to 37.4%±5.2 (n=16) in ZT15 myocytes, (S.E.M; students t-test, P<0.01,) in 100nM ISO. We also looked for any diurnal variation in the arrhythmia threshold to ISO. The percentage of ZT3 myocytes that developed arrhythmias was significantly greater than ZT15 myocytes, with 43.3±4.8% vs. 13.4±1.9% in 3nM ISO, and 61.6±4.4 vs. 35.8±2.7 in 10nM ISO (n=15-18, S.E.M; one-way ANOVA and Bonferroni post hoc test, P<0.01). Our data shows the existence of diurnal variation in E-C coupling, at rest and in response to β-adrenergic stimulation. The data also show diurnal variation in the threshold for arrhythmogenesis to sympathetic stimulation.