Online game playing may induce physiological effects. However, the physical mechanisms that cause these effects remain unclear. The purpose of this study was to examine the physiological effects of long-hour online gaming from an autonomic nervous system (ANS) perspective. Heart rate variability (HRV), a valid and noninvasive electrocardiographic method widely used to investigate ANS balance, was used to measure physiological effect parameters. This study used a five-time, repeated measures, mixed factorial design. Results found that playing violent games causes significantly higher sympathetic activity and diastolic blood pressure than playing nonviolent games. Long-hour online game playing resulted in the gradual dominance of the parasympathetic nervous system due to physical exhaustion. Gaming workload was found to modulate the gender effects, with males registering significantly higher sympathetic activity and females significantly higher parasympathetic activity in the higher gaming workload group.