This study aimed to evaluate workload by detecting Heart Rate Variability (HRV) indexes in a sample of 34 pilots while performing simulated flight exercises. A one-way ANOVA with repeated measures was performed to assess the changes of the physiological measures in five standard maneuvers associated with different workload levels. The results show that all the indexes, but the Low Frequency to High Frequency ratio index (LF/HF), have a well-defined trend between the baseline and the en-route phase and with the three phases takeoff, steady turn, and landing. This study provides evidence of a differentiation among low, medium, and high workload levels using the time, frequencies, and non-linear HRV domains of analysis. These findings support the relevance of HRV indexes for workload evaluation, suggesting the development of non-invasive instruments capable of assessing workload in real-time. Further studies may be conducted to investigate whether the same findings could also be applied to more challenging maneuvers in real working conditions.