Remote work (REMOTE) causes an overlap between working and domestic demands. The study of the cardiac autonomic profile (CAP) by means of heart rate variability (HRV) provides information about the impact of REMOTE on workers’ health. The primary aim was to determine whether CAP, self-perceived stress, environmental and workstation comfort are modified during REMOTE. The secondary aim was to explore how these indices are influenced by individual and environmental work-related factors. Fifty healthy office employees alternating REMOTE and in-office (OFFICE) working were enrolled, rated self-perceived stress, environmental and workstation comfort using a visual analogue scale and performed a 24-h electrocardiogram during REMOTE and OFFICE. Stress was lower (5.6 ± 2.2 vs. 6.4 ± 1.8), environmental comfort higher (7.7 ± 1.9 vs. 7.0 ± 1.5), and the workstation comfort poorer (6.2 ± 1.8 vs. 7.5 ± 1.2) during REMOTE. CAP was similar during REMOTE and OFFICE. CAP was influenced by some work-related factors, including the presence of offspring, absence of a dedicated workspace during REMOTE and number of working hours. All these variables determined a decreased vagal modulation. The working setting seems to impact the levels of perceived stress and comfort, but not the CAP. However, individual and environmental work-related factors reduce cardiac vagal modulation during REMOTE, potentially increasing the risk of developing cardiovascular diseases.