Background: Ivabradine is used to treat tachycardia; unlike atenolol, it does not affect blood pressure or myocardial contractility. This study compared the impact of ivabradine and atenolol on heart rate (HR) and HR variability (HRV) during a 24 h period, feeding and sleeping times, via a Holter electrocardiogram in healthy cats. We hypothesised that ivabradine and atenolol would lower the HRs equally well, even at times of excitement and rest, such as during feeding and sleep; that ivabradine, unlike atenolol, would have an effect on HRV. Methods: Five clinically healthy cats were used in the prospective blinded crossover study receiving 3 days of ivabradine (0.30 mg/kg per os twice daily) followed by atenolol (6.25 mg/cat per os twice daily, range 1.3-2.0 mg/kg) or receiving atenolol followed by ivabradine. A placebo period was initiated before the start of the crossover test, data obtained during that period were used as a baseline (BL). Evaluation parameters included HR and HRV, for the whole 24 h period and for feeding and sleeping times, comparing the effect of ivabradine and atenolol with BL. Results:The HR for the whole 24 h, feeding and sleeping times, were significantly lower with ivabradine and atenolol, compared to BL (p < 0.05). The HRV for the whole 24 h and sleeping time were significantly higher after ivabradine compared with BL and after atenolol. Conclusions: In healthy cats, ivabradine and atenolol significantly reduced the HR regardless of excitement and rest; their effects were comparable. Ivabradine significantly increased HRV in comparison to BL whereas atenolol did not.