BACKGROUND: This study aimed (1) to analyse the effect of non-occupational physical activity (NOPA) on the stress levels of fitness professionals, and (2) to apply a questionnaire to workers measuring burnout syndrome, working conditions and job satisfaction, and to compare the results with physiological stress and recovery measured objectively through heart rate variability (HRV). METHODS: The HRV of 26 fitness instructors was recorded during 2-5 workdays using Firstbeat Bodyguard 2. Participants also completed a questionnaire (CESQT) measuring working conditions and job satisfaction variables and occupational burnout syndrome. RESULTS: NOPA showed a negative association with both the percentage of stress (p < 0.05) and stress-recovery ratio (p < 0.01), and a positive association with the percentage of recovery (p < 0.05). Better work conditions (working hours, salary satisfaction and length of service) were associated with lower stress in fitness professionals. CONCLUSION: NOPA appears to improve the stress levels of fitness instructors in this study cohort. Self-reported burnout levels measured through the CESQT questionnaire do not coincide with the physiological stress responses measured through HRV. Better working conditions appear to reduce the stress response in fitness professionals.