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Breaking prolonged sitting reduces postprandial glucose and insulin concentrations and influences skeletal muscle molecular signaling pathways, but it is unknown whether breaking sitting also affects adipose tissue.Eleven central overweight participants (seven men and four postmenopausal women) 50 ± 5 yr old (mean ± SD) completed two mixed-meal feeding trials (prolonged sitting vs breaking sitting) in a randomized, counterbalanced design. The breaking sitting intervention comprised walking for 2 min every 20 min over 5.5 h. Blood samples were collected at regular intervals to examine metabolic biomarkers and adipokine concentrations. Adipose tissue samples were collected at baseline and at 5.5 h to examine changes in mRNA expression and secretion of selected adipokines ex vivo.Postprandial glycemia and insulinemia were attenuated by approximately 50% and 40% in breaking sitting compared with prolonged sitting (iAUC: 359 ± 117 vs 697 ± 218 mmol per 330 min·L−1, P = 0.001, and 202 ± 71 vs 346 ± 150 nmol per 330 min·L−1, P = 0.001, respectively). Despite these pronounced and sustained differences in postprandial glucose and insulin concentrations, adipose tissue mRNA expression for various genes (interleukin 6, leptin, adiponectin, pyruvate dehydrogenase kinase isozyme 4, insulin receptor substrates 1 and 2, phosphoinositide 3-kinase, and RAC-alpha serine/threonine-protein kinase) and ex vivo adipose tissue secretion of interleukin 6, leptin, and adiponectin were not different between trials.This study demonstrates that breaking sitting with short bouts of physical activity has very pronounced effects on systemic postprandial glucose and insulin concentrations, but this does not translate into corresponding effects within adipose tissue.