Affect is a key determinant of performance, due to its influence on cognitive processing. Negative emotions such as anxiety are recognized cognitive stressors shown to degrade decision making and situation awareness. Conversely, positive affect can improve problem solving and facilitate recall. This exploratory pilot study used electromyography and pupillometry measures to track pilots' levels of negative and positive affect while training in a flight simulator. Fixation duration and saccade rate were found to correspond reliably to pilot self-reports of anxiety. Additionally, large increases in muscle activation were also recorded when higher anxiety was reported. Decreases in positive affect correlated significantly with saccade rate, fixation duration, and mean saccade velocity. Results are discussed in terms of using psychophysiological measures to provide a continuous, objective measure of pilot affective levels as an additional evaluation method to support assessment of pilot performance in simulation training environments.