This study measured the correlation between fatigue and physiological stress symptoms in 102 air traffic controllers (ATCs) in Taiwan. The subjects were asked to complete a fatigue questionnaire and physiological measurement before and after work. The study results showed that nearly 50% of the subjects felt tired and weary after work. However, according to the results of the critical flicker frequency test, all ATCs performed better after work, and the strength of masculine index fingers improved. The systolic and diastolic blood pressure decreased after work. In addition, the levels of urinary 17-hydroxycorticosteroids (17-OHCS) at the pre-shift and post-shift stages were 1.91 ± 1.01 mg/g cr. and 1.50 ± 0.95 mg/g cr., respectively. This study indicated that, while the ATCs’ subjective ratings showed possible work-induced fatigue, it did not affect their physiological response. The findings suggested that ATC is a stressful job, and that complaints regarding excess work stress should be taken seriously. Subsequently the study proposed appropriate intervention strategies to address the identified problems. Relevance to industry: This paper presents the subjective and objective measures of fatigue in ATCs. Recommendations are proposed with respect to potentially viable countermeasures to reduce the impact of fatigue in ATC operations, and achieve a broader understanding of the causal factors for fatigue in ATC.