Background: Health care workers (HCWs) played a key role in preventing and controlling COVID-19. Higher infection risks and intensive work led to occupational burnout for many HCWs, which may affect their lifestyle behaviors and weight. Objective: This study aimed to assess HCWs’ self-rated health status, overweight and obesity rates, lifestyle behaviors, and psychoemotional changes from 2019 to 2022 across China and to analyze the factors associated with changes from underweight or normal weight in 2019 to overweight or obese in 2022. Methods: In this retrospective study, 100 health care institutions were randomly selected from 5 provinces or regions in China. All HCWs who worked in the institutions for at ≥3 years were invited to complete the electronic questionnaire and participate in the online survey from August 1, 2022, to August 31, 2022. Collected data included changes in lifestyle behaviors (dietary habits, physical activity, sleep quality, smoking, alcohol consumption), psychoemotional conditions (persistent stress or recurrent anxiety or depressed mood), health status, and chronic disease control from December 2019 to August 2022. Height and weight in 2019 and 2022 were retrieved from annual physical examination records. Overweight and obesity were defined as 24.0 kg/m2≤BMI<28.0 kg/m2 (overweight) and BMI≥28.0 kg/m2 (obesity). Chi square tests and ANOVAs were used to assess the associations between groups. Logistic regression models were used to analyze the factors associated with HCWs becoming overweight or obese from 2019 to 2022. Results: The questionnaire was submitted by 23,234 HCWs. Of the underweight or normal weight HCWs in 2019, 12.67% (1486/23,234) became overweight or obese in 2022; this change was associated with the following factors: 34-43 years old (OR 0.843, 95% CI 0.740-0.960), 44-53 years old (OR 0.738, 95% CI 0.635-0.960), and 54-63 years old (OR 0.503, 95% CI 0.368-0.685; reference: 24-33 years old), reduction in or never or rarely engaging in physical activity (OR 1.201, 95% CI 1.055-1.368; reference: increase in physical activity; P=.006), increased appetite (OR 2.043, 95% CI 1.788-2.034; reference: reduction or no change in appetite; P<.001). From 2019 to 2022, 51.29% (11,917/23,234) of the respondents experienced increased persistent stress or recurrent anxiety or depressed mood; 44.38% (10,311/23,234) stayed up late more often. Increased persistent stress or recurrent anxiety or depressed mood was associated with physical activity (OR 0.421, 95% CI 0.398-0.447; P<.001) and appetite (OR 1.601, 95% CI 1.483-1.728; P<.001). Conclusions: The pandemic was associated with overweight and obesity for HCWs due to changes in lifestyle behaviors, especially reduced physical activity and increased appetite related to increased persistent stress or recurrent anxiety or depressed mood caused by excessive workload. An integrated approach is needed to address overweight and obesity and lifestyle changes among HCWs by releasing negative psychoemotional conditions through workload reduction in future stressful events.