This study investigated time-on-task effects on heart rate variability (HRV) and its relationship with self-reported and cognitive performance. Data were collected from 19 volunteers aged between 18 and 24 years, who performed a Go/NoGo task for 50 min. The interbeat intervals recording at resting baseline and along with the task were assessed by electrocardiogram NeXus-10 apparatus. HRV data (time and frequency domain), self-reported scores, and cognitive performance were compared along the task. The variables’ reactivity during mental fatigue (fifth minus first block values) were also correlated. Results indicated that time-on-task effects cause a decrease in parasympathetic activity (rMSSD and pNN50), self-report scales (attention, drowsiness, and motivation) and cognitive impairment (RT and error frequency). Only frequency domain (LF/HF) had relation with self-report measures, suggesting a link between the HRV and psychometric measurement of mental fatigue. These results suggest that time-on-task effects on HRV are related to decrease in parasympathetic activity, in which time domain indices have more reliability to monitoring autonomic changes during mental fatigue induction while the frequency domain is related to psychological symptoms of mental fatigue.