Introduction: Modifications of hearth rate variability (HRV) constitute a marker of the autonomic nervous system (ANS) deregulation, a promising pathway linking job strain (JS) and cardiovascular diseases (CVD). The study objective is to assess whether exposures to recent and prolonged JS reduce time-domain HRV parameters on working days (WD) among CVD-susceptible nurses and whether the association also persists on resting days (RD). Material and methods: 313 healthy nurses were investigated twice with one year interval to assess JS based on the demand-control and the effort-reward models. 36, 9 and 16 CVD-susceptible nurses were classified as low JS in both surveys (stable low strain – SLS), recent high JS (high JS at the second screening only-RHS) and prolonged high JS (high strain in both surveys-PHS), respectively. In 9, 7 and 10 of them, free from comorbidities/treatments interfering with HRV, two 24-h ECG recordings were performed on WD and RD. Differences in the time domain HRV metrics among JS categories were assessed using ANCOVA, adjusted for age and smoking. Results: In the entire sample (mean age: 39 years, 83% females) the prevalence of high job strain was 38.7% in the second survey. SDNN (standard deviation of all normal RR intervals) on WD significantly declined among JS categories (p = 0.02), with geometric mean values of 169.1, 145.3 and 128.9 ms in SLS, RHS, PHS, respectively. In the PHS group, SDNN remained lower on RD as compared to the low strain subjects (142.4 vs. 171.1 ms, p = 0.02). Similar findings were found for the SDNN_Index, while SDANN (standard deviation of average RR intervals in all 5 min segments of registration) mean values reduced in the PHS group during WD only. Conclusions: Our findings suggest that persistent JS lowers HRV time-domain parameters, supporting the hypothesis that the ANS disorders may play an intermediate role in the relationship between work stress and CVD.