Presence of heart rate variability (HRV) in isolated hearts is widely recognized; however, its mechanisms are still subject of debate. One of the possible explanations is that mechanoreceptors in cardiac tissue affect HRV. In order to evaluate possible dependence of HRV on heart mechanoreceptors activated during left ventricle filling, the HRV parameters in two perfusion modes of isolated heart were compared: Langendorff and working heart mode. Ten New Zealand rabbit isolated hearts were perfused (Krebs-Henseleit, 37°C, 85 mmHg) in Langendorff mode and consecutively in working heart mode (8 cmH2O preload, 60 cmfyO afterload). A total of 27 HRV parameters in time, frequency, geometric, and non-linear domain were computed. No significant differences (Wilcoxon signed rank test, p < 0.05) were found among all studied HRV parameters between Langendorff and working heart mode. The study confirms the presence of heart rate fluctuation in isolated hearts in both Langendorff and working heart modes. Results of statistical analysis show that heart rate fluctuation is irrespective to mechanical stimulation of heart during atrium and ventricle filling.