Background Many older adults spend the majority of their waking hours sitting, which increases their risk of chronic diseases. Given the challenges that many older adults face when engaging in moderate-to-vigorous physical activity, understanding the health benefits of decreasing sitting time and increasing the number of sit-to-stand transitions is needed to address this growing public health concern. Objective The aim of this 3-arm randomized controlled trial is to investigate how changes in sitting time and brief sit-to-stand transitions impact biomarkers of healthy aging and physical, emotional, and cognitive functioning compared with a healthy attention control arm. Methods Sedentary and postmenopausal women (N=405) will be recruited and randomly assigned to 1 of the 3 study conditions for 3 months: healthy living attention control (Healthy Living), reduce sitting time (Reduce Sitting), and increase sit-to-stand transitions (Increase Transitions). Assessments conducted at baseline and 3 months included fasting blood draw, blood pressure, anthropometric measurements, physical functioning, cognitive testing, and 7 days of a thigh-worn accelerometer (activPAL) and a hip-worn accelerometer (ActiGraph). Blood-based biomarkers of healthy aging included those associated with glycemic control (glycated hemoglobin, fasting plasma insulin and glucose, and homeostatic model assessment of insulin resistance). Results Recruitment began in May 2018. The intervention is ongoing, with data collection expected to continue through the end of 2022. Conclusions The Rise for Health study is designed to test whether 2 different approaches to interrupting sitting time can improve healthy aging in postmenopausal women. Results from this study may inform the development of sedentary behavior guidelines and interventions to reduce sitting time in older adults. Trial Registration ClinicalTrials.gov NCT03473145; https://clinicaltrials.gov/ct2/show/NCT03473145 International Registered Report Identifier (IRRID) DERR1-10.2196/28684