Physical activity impacts positively on stress and anxiety. Working conditions affect the quality of life by increasing stress levels, which can affect job performance and work absence. The Perceived Stress Scale (PSS), Work Ability Index (WAI), Transtheoretical Model (TTM), as well as heart rate variability (HRV) have been applied to monitor the state of workers in their job. The aim of this study was to analyze PSS, WAI, and TTM classifications, and to find out how are they linked to physiological stress (HRV). One hundred and thirteen office workers responded to the three questionnaires and their HRV was monitored for at least two full days. Groups were set up according to TTM (Stage 1, Stage 2, Stage 3-4, Stage 5), WAI (weak WAI, medium WAI, good WAI), and PSS (low PSS, medium PSS, high PSS). Results obtained from the test were related to stress values measured by HRV with a Bodyguard2 device. The Stage 5 group from TTM had better HVR and stress levels than the other groups for both women and men (p < 0.05). Participants in the good WAI group and low PSS group had better results than weak WAI and high PSS, but the differences with respect to medium WAI and medium PSS were less clear. Finally, TTM seemed to be the best tool to discriminate physiological stress in office workers with regard to other questionnaires.