Studies have shown that physical activity influences affective states. However, studies have seldom depicted these associations in ongoing real-life situations, and there is no investigation showing that motivational states (i.e., more or less autonomously regulated) would moderate these effects in situ. To investigate the interaction of autonomous regulation and actual physical activity (aPA) with affective states, we use an ambulatory assessment approach. The participants were 44 university students (mean age: 26.2 ± 3.2 years). We assessed aPA through 24-hr accelerometry and affective states and autonomous regulation via electronic diaries. Palmtop devices prompted subjects every 45 min during a 14-hr daytime period. We performed hierarchical multilevel analyses. Both aPA and autonomous regulation significantly influenced affective states. The interaction was significant for two affects. The higher the volume of aPA and thereby the more autonomously regulated the preceding bout of aPA was, the more our participants felt energized (r = .16) but agitated (r = -.18).