Background Sedentary behavior (SB) is a biologically distinct yet understudied cardiovascular disease risk (CVD) factor. However, specific public health policy regarding the optimal strategy for SB interruption is unavailable. This paper outlines the protocol for part I of the Sitting with Interruption and Whole-Body Cardiovascular Health (SWITCH) study, including the rationale, objectives, methodology, and next steps. We additionally detail practical considerations that went into the development of the NIH R01 grant supporting this research. Methods Healthy men and women (n = 56, aged 36–55) who are inactive (textless90 min/wk. of moderate-to-vigorous intensity physical activities for past 3 months) and sedentary (sitting for textgreater8 h/day), will be recruited for this randomized crossover trial. Specifically, participants will complete the following 4-h conditions: (i) SB with once/h 5 min walk break; (ii) SB with once/h 15 min stand break; (iii) SB with twice/h breaks (alternating 5 min walk and 15 min stand); and (iv) SB with no breaks (i.e., control). Focus group discussions will refine our socioecological SB reduction model. Results The primary outcome will be change in aortic arterial stiffness (i.e., pulse wave velocity; PWV, m/s) for each substitution strategy relative to the control (SB with no breaks) condition. Conclusions The outcomes from this study will facilitate the design of a subsequent randomized controlled trial to test a mechanism-informed, feasible SB-reduction intervention and support the development of SB policy.