Abstract Background In July, 2019, the World Health Organization (WHO) commenced work to update the 2010 Global Recommendations on Physical Activity for Health and established a Guideline Development Group (GDG) comprising expert public health scientists and practitioners to inform the drafting of the 2020 Guidelines on Physical Activity and Sedentary Behavior. The overall task of the GDG was to review the scientific evidence and provide expert advice to the WHO on the amount of physical activity and sedentary behavior associated with optimal health in children and adolescents, adults, older adults (> 64 years), and also specifically in pregnant and postpartum women and people living with chronic conditions or disabilities. Methods The GDG reviewed the available evidence specific to each sub-population using systematic protocols and in doing so, identified a number of gaps in the existing literature. These proposed research gaps were discussed and verified by expert consensus among the entire GDG. Results Evidence gaps across population sub-groups included a lack of information on: 1) the precise shape of the dose-response curve between physical activity and/or sedentary behavior and several of the health outcomes studied; 2) the health benefits of light-intensity physical activity and of breaking up sedentary time with light-intensity activity; 3) differences in the health effects of different types and domains of physical activity (leisure-time; occupational; transportation; household; education) and of sedentary behavior (occupational; screen time; television viewing); and 4) the joint association between physical activity and sedentary time with health outcomes across the life course. In addition, we acknowledge the need to conduct more population-based studies in low- and middle-income countries and in people living with disabilities and/or chronic disease, and to identify how various sociodemographic factors (age, sex, race/ethnicity, socioeconomic status) modify the health effects of physical activity, in order to address global health disparities. Conclusions Although the 2020 WHO Guidelines for Physical Activity and Sedentary Behavior were informed by the most up-to-date research on the health effects of physical activity and sedentary time, there is still substantial work to be done in advancing the global physical activity agenda.