CONTEXT: Postprandial dysmetabolism in type 2 diabetes (T2D) is exacerbated by prolonged sitting and may trigger inflammation and oxidative stress. It is unknown what impact countermeasures to prolonged sitting have on the postprandial lipidome. OBJECTIVE: In this study, we investigated the effects of regular interruptions to sitting, compared with prolonged sitting, on the postprandial plasma lipidome. DESIGN: Randomized crossover experimental trial. SETTING: Participants underwent three 7-hour conditions: uninterrupted sitting (SIT); light-intensity walking interruptions (LW); and simple resistance activity interruptions (SRA). PARTICIPANTS AND SAMPLES: Baseline (fasting) and 7-hour (postprandial) plasma samples from 21 inactive overweight/obese adults with T2D were analyzed for 338 lipid species using mass spectrometry. MAIN OUTCOME MEASURES: Using mixed model analysis (controlling for baseline outcome variable, gender, body mass index, and condition order), the percentage change in lipid species (baseline to 7 hours) was compared between conditions with Benjamini-Hochberg correction. RESULTS: Thirty-seven lipids were different between conditions (P < 0.05). Compared with SIT, postprandial elevations in diacylglycerols, triacylglycerols, and phosphatidylethanolamines were attenuated in LW and SRA. Plasmalogens and lysoalkylphosphatidylcholines were reduced in SIT, compared with attenuated reductions or elevations in LW and SRA. Phosphatidylserines were elevated with LW, compared with reductions in SIT and SRA. CONCLUSION: Compared with SIT, LW and SRA were associated with reductions in lipids associated with inflammation; increased concentrations of lipids associated with antioxidant capacity; and differential changes in species associated with platelet activation. Acutely interrupting prolonged sitting time may impart beneficial effects on the postprandial plasma lipidome of adults with T2D. Evidence on longer-term intervention is needed.