Background/aim Anesthetics are often used in animal experiments to achieve immobilization and relieve pain. However, many anesthetics can alter the dynamics of cardiovascular systems. We aimed to compare the effects of two frequently used anesthetics agents on heart rate variability (HRV) parameters in mice. Materials and methods This observational study was performed between May and June 2014 in 21 male BALB/c mice aged 16–20 weeks. The animals were divided into three groups: pentobarbital (P), (n = 7); pentobarbital+fentanyl (P+F), (n = 7); and ketamine+xylazine (K+X), (n = 7). Surface electrocardiography (ECG) electrodes were placed in lead II configuration. The tachogram of RR intervals was obtained after R waves were detected using the Pan-Tompkins real-time QRS recognition algorithm. Frequency-domain, time-domain, and nonlinear HRV analyses were performed. Results The bradycardia effect was higher in the K+X group (p < 0.01). Time-domain indices were higher in group K+X compared to group P (p < 0.01) and group P+F (p < 0.001). Very low frequency (VLF) power was significantly lower in group K+X compared to group P and group P+F (p < 0.01). Low frequency (LF) power, low frequency/high frequency (LF/HF) ratio, and total power (TP) were higher in group K+X compared to group P (p < 0.01) and group P+F (p < 0.001). The detrended fluctuation analysis short-term parameter (DFAα 1 ) was significantly higher in group K+X compared to group P+F (p < 0.05) and the long-term parameter (DFAα 2 ) was lower in group K+X compared to group P (p < 0.05). Standard deviations SD1 and SD2 were higher in group K+X compared to group P (p < 0.001) and group P+F (p < 0.001), SD2/SD1 ratio was lower in group K+X compared to group P (p < 0.05) and group P+F (p < 0.05). Entropy measures did not differ between groups. Conclusion HRV analyses, including nonlinear methods, indicated that a K+X combination reduces imbalance and disorder in the regulation of the autonomic nervous system (ANS) in comparison to both P and the P+F combination.