Background <p>Heart rate variability (HRV) provides insight into cardiac autonomic regulation, while nonlinear HRV indices may capture dynamic features of cardiac rhythm that are not fully reflected by conventional time- and frequency-domain parameters. Flecainide and isoproterenol influence cardiac rhythm through different mechanisms, sodium-channel blockade and β-adrenergic stimulation, respectively. This study investigated whether these pharmacological perturbations alter HRV and RR interval fractal organization in mice.</p> Methods <p>Twenty-four adult male mice were assigned to control (CNT, <i>n</i> = 9), flecainide (FLE, <i>n</i> = 8), or isoproterenol (ISO, <i>n</i> = 7) groups. Electrocardiographic recordings were obtained under anesthesia before and after drug administration. HRV analysis was performed using RR interval series. Time-domain, frequency-domain, Poincaré plot, and detrended fluctuation analysis (DFA) parameters were calculated. Fractal complexity loss was assessed using DFA-derived area-based indices, including dS1, dS2, and total deviation score (TdS). Pre- and post-drug values were compared using a two-way mixed-design ANOVA (Group × Time) with Šídák-corrected post-hoc tests.</p> Results <p>A significant Group × Time interaction was observed for mean heart rate (<i>p</i> &lt; 0.001) and mean RR interval (<i>p</i> = 0.013). Post hoc analyses showed that isoproterenol markedly increased heart rate from 391 to 548 BPM (<i>p</i> = 0.002), whereas flecainide produced no significant change in heart rate. DFAα1 also demonstrated a strong Group × Time interaction (<i>p</i> &lt; 0.001), increasing after isoproterenol but decreasing after flecainide. All DFA-derived complexity-loss indices showed significant Group × Time interactions, including dS1 (<i>p</i> &lt; 0.001), dS2 (<i>p</i> = 0.002), and TdS (<i>p</i> = 0.001). Flecainide increased fractal complexity loss, whereas isoproterenol reduced it; accordingly, post-drug complexity-loss values were higher in the flecainide group than in both the isoproterenol and control groups. In contrast, conventional HRV magnitude-based indices, including SDNN, spectral power measures, stress index, and DFAα2, did not show significant Group × Time interactions.</p> Conclusion <p>Flecainide and isoproterenol produced distinct alterations in cardiac rhythm dynamics in anesthetized mice. DFAα1 and DFA-derived area-based indices, particularly dS1 and TdS, provided complementary information about drug-induced changes in RR interval fractal organization. These findings should be interpreted as exploratory evidence of altered rhythm organization rather than as diagnostic evidence of specific arrhythmia types or cardiac health status.</p> Graphical Abstract <p></p>