Objective: Just-in-time adaptive interventions (JITAIs) use mobile computers, sensors, and software analytics to automatically detect behavior and deliver tailored treatment. However, little is known about how JITAIs influence patterns of behavior or how best to design JITAIs for maximum effect. Method: This study examined prompts and behavioral response to the B-MOBILE JITAI for reducing sedentary behavior (SB) in overweight/obese individuals. Thirty participants (83% women; 67% White, mean ± SD body mass index = 36.2 kg/m2) tested 3 conditions presented in a randomized counterbalanced order involving smartphone-based prompts for walking breaks of (a) 3 min after 30 SB min, (b) 6 min after 60 SB min, and (c) 12 min after 120 SB min. Results: Participants carried the smartphone an average of 6.90 days during each 7-day condition, for an average of 14.94 hr per day. The 3- and 6-min conditions resulted in the greatest number of prompts, walking breaks, the best adherence to prompts, the greatest amount of daily time spent in walking breaks, and fastest adherence to prompts (ps < .01). Small but statistically significant decreases in the number of daily walking breaks, adherence to prompts, and minutes per day spent in walking breaks were observed as a function of the number of days spent in a condition (ps < .05). Conclusions: The B-MOBILE JITAI was effective in prompting breaks in sedentary behavior when it was most clinically relevant. Frequent prompts for small change may be an optimal strategy for shaping sedentary behavior, although more research is needed to determine how best to promote long-term adherence.