Understanding the complex relationship between sedentary breaks, affective well-being, and cognition in daily life is critical as modern lifestyles are increasingly characterized by sedentary behavior. Previous studies have provided evidence that short sedentary breaks are feasible to integrate into daily life and can improve affective and cognitive parameters. However, observational studies do not allow for causal interpretation. To overcome this limitation, we conducted the first empirical study that integrated the within-person encouragement approach to test the causal effects of short 3-minute sedentary breaks on affective and cognitive parameters in daily life. We recruited 222 university employees in a two-weeks ambulatory assessment study. In particular, physical behavior was captured with a thigh-worn accelerometer while completing up to six smartphone-based assessments of self-reported affective states and working memory performance. Results of two-level structural equation modeling revealed that compared to uninterrupted sedentary bouts (≥ 30 minutes), short three-minute sedentary breaks had a positive causal effect on the affective state dimensions valence and energetic arousal. Contrary to expectations, no significant effect was found for working memory performance and the affective state dimension of calmness. Furthermore, as expected the variation in encouragements regarding the intensity level (i.e., standing, slow walking, and fast walking) positively predicted the following intensity behavior, and the intensity rating was linearly associated with valence and energetic arousal. Our methodological approach demonstrated the possibility of moving towards investigating causal effects in everyday life showing that short sedentary breaks are positively related to affective states.