Abstract Air traffic controllers (ATCOs) are exposed to high workload even during routine operations. Their job carries a lot of responsibility and consists of complex tasks. In order to enhance high safety standards, a systematic evaluation of workload levels is required. This study explores an objective measurement of workload. Heart activity of ATCOs was monitored in order to indicate psychological status of ATCOs while being exposed to different levels of workload - in terms of changing traffic loads and airspace complexity. A standard method was used for the evaluation of heart activity. This method includes the frequency analysis in context of heart rate variability; time intervals of consecutive R waves in the heart activity record are assessed. The evaluation of the ratio of power in the low-frequency and high-frequency bands (indicator of sympathovagal balance) as well as parameters derived from fluctuation analysis were used. The results indicated that parameters of heart rate variability based on spectral analysis were significant in case of workload evaluation.