The interaction of prolonged sitting with physical exercise for maintaining health is unclear. We tested the hypothesis that prolonged siting would have a deleterious effect on postprandial plasma lipemia (PPL, postprandial plasma triglycerides) and abolish the ability of an acute exercise bout to attenuate PPL. Seven healthy young men performed three interventions over 5 days (D1-5) in a randomized crossover design with > 1 week between interventions: 1) sitting >14h/d with hypercaloric energy balance (SH), 2) sitting >14h/d with net energy balance (SB), and 3) active walking/standing with net energy balance (WB) and sitting 8.4 h/d. The first high fat tolerance test (HFTT1) was performed on D3 following 2 days of respective interventions. On the evening of D4 subjects ran on a treadmill for 1-h at ~ 67% VO_2max, followed by the second HFTT (HFTT2) on D5. Two days of prolonged sitting increased TG AUCI (i.e., incremental area under the curve for TG), irrespective of energy balance, compared to WB (27% in SH, p=0.003 and 26% in SB, p=0.046). Surprisingly, after four days of prolonged sitting (i.e.; SH and SB), the acute exercise on D4 failed to attenuate TG AUCI or increase relative fat oxidation in HFTT2, compared to HFTT1, independent of energy balance. In conclusion, prolonged sitting over several days was sufficient to amplify PPL and to abolish the beneficial effect of acute exercise on lowering PPL and raising fat oxidation, regardless of energy balance. This underscores the importance of limiting sitting time even in people who have exercised.