Background: Providing feedback is a technique to promote health behavior that is emphasized by behavior change theories. However, these theories make contradicting predictions regarding the effect of the feedback sign—that is, whether the feedback signals success or failure. Thus, it is unclear whether positive or negative feedback leads to more favorable behavior change in a health behavior intervention. Objective: The aim of this study was to examine the effect of the feedback sign in a health behavior change intervention. Methods: Data from participants (N=1623) of a 6-month physical activity intervention was used. Participants received a feedback email at the beginning of each month. Feedback was either positive or negative depending on the participants’ physical activity in the previous month. In an exploratory analysis, change in monthly step count averages was used to evaluate the feedback effect. Results: The feedback sign did not predict the change in monthly step count averages over the course of the intervention (b=−84.28, P=.28). Descriptive differences between positive and negative feedback can be explained by regression to the mean. Conclusions: The feedback sign might not influence the effect of monthly feedback emails sent out to participants of a large-scale physical activity intervention. However, randomized studies are needed to further support this conclusion. Limitations as well as opportunities for future research are discussed.