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Purpose: Sedentary time relates to higher anxiety and more negative affect in children. This study assessed whether interrupting sitting over 3 hours is sufficient to influence state anxiety, positive affect, or negative affect, and tested weight status as a moderator., Methods: Analyses were the second (preplanned) purpose of a larger study. Children (N = 61; age: mean [SD] = 9.5 [1.3]; 43% healthy weight) completed 2 experimental conditions: continuous sitting for 3 hours and sitting for 3 hours interrupted with walking for 3 minutes in every 30 minutes. State anxiety, positive affect, and negative affect were reported at pretest and posttest. Multilevel models for repeated measures assessed whether experimental condition predicted posttest scores., Results: Experimental condition was unrelated to posttest state anxiety or positive affect. Weight status moderated how experimental condition influenced posttest negative affect (P = .003). Negative affect was lower in the children of healthy weight after interrupted sitting (vs continuous sitting; β = -0.8; 95% confidence interval, -1.5 to 0.0, P = .05), but it was higher in the children with overweight/obesity after interrupted sitting (vs continuous sitting; β = 0.6; 95% confidence interval, 0.0 to 1.2, P = .06)., Conclusions: Interrupting sitting acutely reduced negative affect in children of healthy weight, but not in children with overweight. Further research is needed to better understand the potential emotional benefits of sitting interruptions in youth.