ABSTRACT Sedentary behaviors (SB) are biologically distinct but understudied cardiovascular disease (CVD) risk factors. National and international agencies have surmised that the level of evidence for an overall and dose-response association between SB and CVD mortality is moderate-strong. These agencies do not provide specific recommendations for breaking-up SB, though do call on the research community to facilitate policy development by establishing biological plausibility, identifying the optimal dose for SB substitution strategies, and conducting randomized clinical trials to test the efficacy of these strategies. Our goal is to identify mechanism-informed, socioecological-based SB substitution recommendations to mitigate CVD risk. Aim 1A will identify a feasible SB substitution strategy to prevent the adverse cardiovascular responses to prolonged SB. Adverse cardiovascular responses will be measured using aortic arterial stiffness (AS), a noninvasive test that predicts future CVD. To accomplish this aim, in 56 middle-aged (36-55 years) participants, we will measure aortic AS and associated mechanistic responses to the following over 4 hours: (i) SB with once/hour 5 min walk break; (ii) SB with once/hour 15 min stand break; (iii) SB with twice/hour breaks (alternating 5 min walk and 15 min stand); and (iv) SB with no breaks (control). These strategies were selected based on extensive prior work by our group, and because they are feasible, which is a key component of this proposal. SB reduction strategies will only decrease CVD risk if people are willing to adhere to future SB substitution recommendations. To increase the likelihood of feasibility and long-term adherence, Aim 1B will evaluate the determinants of SB using a socioecological model. This recognizes that behavior change is likely to be limited if the physical and sociocultural environments do not support the behavior change. To accomplish Aim 1B, a combined inductive-deductive qualitative approach will be used. Participants who complete Aim 1A will be participate in one of 6 focus groups (6-8 participants/group). Aim 2A will then test the feasibility of implementing the strategy in the real-world via an individualized single-arm 12-week intervention (n=40, 36-64 years). The mechanistic (Aim 1A) and qualitative (Aim 1B) data from Aim 1 will guide the intervention design. We will test the following feasibility domains: demand, acceptability, implementation, practicality, integration, and limited-efficacy. Lastly, Aim 2B will investigate which components of the intervention worked and which components did not work. All participants who complete Aim 2A will be asked to complete questionnaires pertaining to our behavioral model as well as participate in a focus group. The focus groups will be used to add context to the quantitative feasibility data. Specifically, we will address acceptability, adoption, appropriateness, sustainability, and the perceived socioecological determinants. Cru...