To determine whether interrupting prolonged sitting with three different activity breaks has both acute and chronic effects on postprandial lipid metabolism immediately after the activity‐break period and on the following day, this study is a secondary analysis of an experimental research, which included 16 sedentary healthy adults (7 men, 24 ± 3 years, BMI 22.2 ± 2.3 kg/m2) who completed four 26‐h laboratory trials. Participants spent 22.5 hours in a whole room calorimeter for testing energy expenditure (EE), including a 9‐h activity‐break period: (a) 9‐h prolonged sitting (SIT); (b) 3 minutes of brisk walking (60% VO2max) in between every 30‐min sitting bout (WALK3), (c) 5 minutes every 45‐min (WALK5), and (d) 8 minutes every 60‐min (WALK8). Total area under the curve (tAUC) and incremental AUC (iAUC) for 2‐h postprandial serum triglyceride (TG) levels and non‐esterified fatty acid (NEFA) levels were examined immediately after the 9‐h trial (post‐dinner) and the next morning (post‐breakfast). WALK8 reduced the post‐breakfast TG tAUC by 11% (P = .041) relative to SIT, and the effect was attenuated after adjustment for EE. The tAUC and iAUC indicated no significant treatment effects on post‐dinner TG and NEFA, and post‐breakfast NEFA in any of the activity‐break trials. EE was positively associated with the post‐breakfast NEFA iAUC (unstandardized β = 0.17 µmol/L/MJ [0.05‐0.28], P = .006). There was a chronic effect of interrupting sitting with short bouts (8 minutes) of brisk walking every 60 minutes on postprandial lipemia the following morning after intervention, and higher activity bout‐induced EE may be more effective in sedentary, healthy adults.