Pilots in long-duration flight missions in single-seat aircraft may be affected by fatigue. This study determined associations between cognitive performance, emotions and physiological activation and deactivation–measured by heart rate variability (HRV)–in a simulated 11-h flight mission in the 39 Gripen aircraft. Twelve participants volunteered for the study. Perceived fatigue was measured by the Samn-Perelli Fatigue Index (SPFI). Cognitive performance was measured by non-executive and executive tasks. Emotions were assessed by the Circumplex Affect Space instrument. HRV was considered in relation to the cognitive tasks in four time points–Hours 3, 5, 7, 9–and their associations with emotional ratings. Results indicated a decrease in performance in the non-executive task after approximately 7 h. This result was correlated with self-reported measures of fatigue. HRV, assessed by indices of parasympathetic modulation, remained unchanged for both non-executive and executive tasks over time (p >.05 for all). Significant correlations were observed between emotions and HRV; with increased boredom, increased passiveness, decreased stimulation, and decreased activeness, HRV indicators increased (p <.05). This suggests that a low self-regulatory effort for maintaining performance in these conditions was prevalent and that pilots could adapt to some degree to the demands and fatigue of long-duration missions.