The work schedules of airline crewmembers include extended workdays, compressed work periods, and limited time for recovery, which may lead to cardiovascular strain and fatigue. The aim of this study was to evaluate changes in heart rate variability (HRV) during work and sleep, and with respect to work characteristics and breaks. We followed 49 airline crewmembers during four consecutive workdays of ≥39 h. Data included HRV measurements, a questionnaire, and sleep/work diaries. HRV parameters include root mean square of successive differences (RMSSD), standard deviation of the normal beat-to-beat differences (SDNN), and the low and high frequency ratio (LF/HF). The results indicate higher levels of cardiovascular strain on the 4th compared to the 1st workday, most prominent among cabin crewmembers. In this group, we observed indications of decreased cardiovascular strain by increasing duration of sleep, demonstrated by increased RMSSD (B = 2.7, 95% CI 1.6, 3.8) and SDNN (B = 4.4, 95% CI 3.0, 5.7), and decreased LF/HF (B = -0.2, 95% CI, -0.4,-0.01). Similarly, longer duration of breaks was associated with lower cardiovascular strain, indicated by increased RMSSD (B = 0.1, 95% CI 0.03, 0.1) and SDNN (B = 0.1, 95% CI 0.1, 0.1). Among pilots, increased LF/HF indicated higher cardiovascular strain in those who often or always reported of high workload (B = 4.3, 95% CI 2.3, 6.3; and B = 7.3, 95% CI 3.2, 11.4, respectively). The results support the contention that the studied work period increases cardiac strain among airline crew. Work characteristics, breaks, and sleep are associated with changes in HRV.