Introduction: According to earlier research, fitness training can reduce the cardiovascular risks of waterpipe tobacco use. This study aimed to investigate how electrocardiogram (ECG) parameters and heart rate variability (HRV) changes occur in Wistar rats exposed to continuous hookah smoke and moderate endurance exercise training. Methods: 28 adult male Wistar rats, weighing 180–220 g, were divided into 4 groups as control group (CTL), exercise training group (Ex), waterpipe tobacco smoke exposure group (WPS), andexercise training and waterpipe tobacco smoke group (WPS+Ex). After completing the 8-week smoke exposure and exercise protocol, ECG recording, HRV characteristics, including frequency domains, temporal domains, and non-linear analysis, were computed. Results: The findings showed that, compared to the control group, hookah smoking significantly increased the RR interval (P < 0.05) and decreased heart rate (HR) and standard deviation of the second heart rate variation (SD2), an indicator of long-term HRV variability. Compared to the CTL group, exercise training decreased the low frequency/high frequency (LF/HF) band non-significantly and increased the HF band while decreasing the LF band (P<0.05). Following exposure to hookah smoking and exercise training, the WPS+Ex group’s SD2 rose (P<0.05), whereas the WPS group’s HF and corrected QT (QTc) interval decreased. Conclusion: Overall, our results demonstrated that 8 weeks of moderate-intensity endurance training had a slight favorable impact on the rats’ HRV frequency domains. At the same time, hookah smoking inhibited this beneficial effect and lowered HR.