Background: The autonomic nervous system plays a curical role in the regulation of vital physiological functions, including cardiac rhythm, heart rate, and maintenance of blood pressure homeostasis. Previous studies have reported that autonomic nervous function gradually declines with aging, particularly after the age of 40, with an estimated reduction of 5-8% per decade. Globally, the prevalence of atrial fibrillation is rapidly increasing, and it is estimated that by 2050, approximately 72 million people in Asia will be diagnosed with atrial fibrillation, with nearly 3 million cases expected to develop ischemic stroke as a complication. Several risk factors, including arterial hypertension, obesity, alcohol consumption, and smoking, contribute to the development of atrial fibrillation, partly by disrupting autonomic nervous system regulation. Patients with atrial fibrillation have a 1.5-2-fold higher risk of mortality compared with the general population, and the lifetime risk of developing atrial fibrillation after the age of 40 is estimated to be approximately 25%. Aim: To assess autonomic nervous system dysregulation based on heart rate variability parameters among individuals aged 40 years and older. Materials and Methods: The stady was hospital-based case–control study was conducted among patients who received outpatient care and underwent 24-hour Holter electrocardiography (ECG) at Promedica Cardiology Hospital between October 2025 and February 1, 2026. A total of 70 participants were selected using a random sampling method. Of these, 31 patients diagnosed with paroxysmal atrial fibrillation were included in the case group, based on clinical records and Holter ECG findings. The control group consisted of 39 participants without atrial fibrillation. Autonomic nervous system regulation was evaluated using time-domain HRV parameters (rMSSD, pNN50) and frequency-domain parameters (LF, HF, and LF/HF ratio). Statistical analyses were performed using STATA version 15.0 A p-value of <0.05 was considered statistically significant. Result: Among the study participants, 37.1% (n=26) were male and 62.9% (n=44) were female. The mean age was 56.9±11 years (range: 40–79). Of the total participants, 44.2% (n=31) belonged to the paroxysmal atrial fibrillation group, while 55.8% (n=39) were included in the control group. A statistically significant difference in age and sex distribution was observed between the two groups (p=0.001). A significant association was identified between autonomic nervous system dysregulation and paroxysmal atrial fibrillation (p=0.001). In the paroxysmal atrial fibrillation group, increased pNN50, rMSSD values, and a decreased LF/HF ratio were observed, indicating impaired parasympathetic (vagal) activity. These alterations were associated with an increased risk of atrial fibrillation, with an odds ratio (OR) of 4.90 (95% CI: 1.98-12.14). No statistically significant differences in autonomic nervous system parameters were observed in the control group. Conclusion Autonomic nervous system dysfunction assessed through heart rate variability parameters may play a significant role in the development of atrial fibrillation. Holter ECG–based heart rate variability analysis could therefore be utilized as a clinical tool for early detection of paroxysmal atrial fibrillation, risk assessment, improved monitoring, and optimization of treatment strategies.