Heart Rate Variability (HRV) has been suggested as a useful tool to assess fatigue-sensitive psychological operations. The present study uses a between and within-subject design with a cognitively demanding task and a documentary viewing condition, to examine the temporal profile of HRV during reactivity, Time-on-Task (ToT), and recovery. In the cognitive task group, participants worked on a bimodal 2-back task with a game-like character (the Gatekeeper task) for about 1.5 hours, followed by a 12-minute break, and a post-break block of performance (about 18 min). In the other group, participants watched documentaries. We hypothesized an increasing vagal-mediated HRV as a function of Time spent on the Gatekeeper task and no HRV change in the documentary viewing group. We also analyzed the trial-based post-response cardiac activity as a physiological associate of task-related motivation. Relative to the documentary-viewing, ToT was associated with an elevated level of subjective fatigue, decreased heart rate, and increased HRV, particularly in the vagal-mediated components. Based on fatigued participants' post-error cardiac slowing, and post-error reaction time analyses, we found no evidence for motivation deficits. The present findings suggest that the parasympathetic branch of the autonomous nervous system functioning as a relaxation system tends to be activated under increasing mental fatigue. In addition, the study shows that many HRV indices also seem to change when individuals are engaged in a prolonged, less fatiguing activity (e.g. documentary viewing). This finding emphasizes the relevance of comparisons/control conditions in ToT experiments.