Fatigue is a major contributing factor to operational errors. Therefore, the validation of objective and sensitive indices to detect fatigue is critical to prevent accidents and catastrophes. Whereas tests based on saccadic velocity (SV) have become popular, their sensitivity in the military is not yet clear, since most research has been conducted in laboratory settings using not fully validated instruments. Field studies remain scarce, especially in extreme conditions such as real flights. Here, we investigated the effects of real, long flights on SV. We assessed five newly commissioned military helicopter pilots during their aviation training. Pilots flew Sikorsky S-76C helicopters, under instrumental flight rules, for more than 2 h (ca. 150 min). Eye movements were recorded before and after the flight with an eye tracker using a standard guided-saccade task. We also collected subjective ratings of fatigue. SV significantly decreased from the Pre-Flight to the Post-Flight session in all pilots by around 3% (range: 1-4%). Subjective ratings showed the same tendency. We provide conclusive evidence about the high sensitivity of fatigue tests based on SV in real flight conditions, even in small samples. This result might offer military medical departments a valid and useful biomarker of warfighter physiological state.