Background: Population studies gathering measured data on fitness and physical behavior, covering physical activity, standing, sedentary behavior, and time in bed, are scarce. This article describes the protocol of the FINFIT 2021 study that measures fitness and physical behavior in a population-based sample of adults and analyzes their associations and dose–response relationships with several health indicators. Methods: The study comprises a stratified random sample of 20–69-year-old men and women (n = 16,500) from seven city-centered regions in Finland. Physical behavior is measured 24/7 by tri-axial accelerometry and analyzed with validated MAD-APE algorithms. Health and fitness examinations include fasting blood samples, measurements of blood pressure, anthropometry, and health-related fitness. Domains of health, functioning, well-being, and socio-demographics are assessed by a questionnaire. The data are being collected between September 2021 and February 2022. Discussion: The study provides population data on physical fitness and physical behavior 24/7. Physical behavior patterns by intensity and duration on an hour-by-hour basis will be provided. In the future, the baseline data will be assessed against prospective register-based data on incident diseases, healthcare utilization, sickness absence, premature retirement, and death. A similar study will be conducted every fourth year with a new random population sample.