Health care professionals are exposed to the psychological and physiological effects of stress, which is a well-known risk factor for various mental and physical health problems. The aims of this study were to assess the adherence of female health care workers to use a web-based tool for improving and modifying lifestyle and to identify the potential factors influencing their adherence. A prospective, observational study was performed. A total of 80 female health care workers (physicians and gradated nurses) from 2 university medical centers and female members of a family medicine society participated. Participants completed a questionnaire that inquired about their basic demographic data and physical fitness. Physical fitness was assessed by the Rockport Fitness Walking Test. Adherence to a web-based application (24@life) was followed for 3 months and the number of log-ins into the application was counted. The study was conducted from March to October 2019. Significantly high workload has been detected in all groups (P<.05), except in the general practitioner with normal workload group. The graduated nurse working in the surgery room group showed chronic stress with elevated S-cortisol levels (>690 nmol/L); activated cellular immune system with elevated concentrations of lymphocytes (reference 1.1-2.5 × 10 cells/L), CD3 cells (reference 0.7-1.9 × 10 cells/L), CD8 cells (reference 0.2-0.7 × 10 cells/L), and HLA-DR/CD3 cells (reference 0.04-0.2 × 10 cells/L); and the worst quality of sleep (mean 2.8 SD 1.2). Only 32 of 80 participants (40%) were adherent to the web-based application. Participants most frequently viewed web pages on areas of physical activity (497 times) and nutrition (332 times). No factors or participant's characteristics such as weight (odds ratio OR 1.026, 95% CI 0.977-1.078), BMI (OR 0.993, 95% CI 0.834-1.184), age (OR 0.970, 95% CI 0.910-1.034), or stress level (OR 0.997, 95% CI 0.995-1.000) were identified to affect the adherence rates. Female health care workers exposed to high workload did not find the web-based application useful for improving and modifying their lifestyle. Therefore, other strategies that might help health care workers facing stress and improve their lifestyle should be identified.