Smoking causes sympathovagal changes and peripheral vessel injury, which can be assessed by heart rate variability (HRV) that includes endothelial nitric oxide (NO) and sLOX-1 markers, respectively. This study aimed to investigate the HRV and NO changes in acute smoking and sLOX-1 overproduction in chronic smoking. A total of 60 males were recruited. Thirty smokers and 30 non-smokers were studied. At first the HRV parameters were obtained from all subjects. Only smokers were then consecutively recorded for 5 min and 15 min after smoking a single cigarette with simultaneous blood collection at each time point for NO and sLOX-1 measurements. We found a significant lowering of HRV and a positive correlation between sLOX-1 and mean arterial pressure in smokers. After 5 min of smoking, an immediate adverse effect of autonomic function was represented by the HRV along with an immediate lowering of NO which is a vasodilator. Acute smoking influences the autonomic neural control from NO-mediated modulation which can be assessed by the noninvasive technique of HRV analysis.