Experimental evidence shows that breaking-up sedentary behavior is positively associated with positive mental health outcomes. However, whether sedentary breaks influence mood in everyday life is largely unknown. Moreover, it is unclear which break patterns are most beneficial to enhance mood.To investigate the degree to which sedentary break patterns influence mood dimensions in everyday life, we conducted an Ecological Momentary Assessment (EMA) study in 92 university employees over 5 days. We continuously measured physical behavior (physical activity and sedentary behavior) objectively via accelerometers and assessed mood 10 times a day on smartphone diaries. We defined distinct break patterns, such as variations in frequency (number of breaks), duration (length of breaks), intensity (metabolic equivalent) and context (home or work), and used multilevel modeling to analyze the within-subject effects of sedentary break patterns on mood.Break intensity was positively associated with subsequent valence (P< 0.01), energetic arousal (P< 0.01) and calmness (P< 0.05). Break frequency was positively associated with subsequent valence and energetic arousal (all Ps < 0.01), but break duration was not associated with mood. Exploratory analyses indicated that breaking-up sedentary behavior was more beneficial at home than at work.These ecologically valid findings suggest breaking-up sedentary behavior as a promising strategy to enhance mood in everyday life. In particular, breaking-up sedentary behavior frequently and intensively, for example by walking instead of standing, may be most beneficial. We call for future studies to substantiate these accounts and to identify both practical and optimal break patterns among different samples. This evidence informs official public health recommendations aiming to "minimize sedentary time in everyday life".