Prolonged periods of sedentary behavior are linked to cardiometabolic disease independent of exercise and physical activity. This study examined the effects of posture by comparing one day of sitting (14.4 ± 0.3 h) to one day of standing (12.2 ± 0.1 h) on postprandial metabolism the following day. Eighteen subjects (9 men, 9 women; 24 ± 1 y) completed two trials (sit or stand) in a crossover design. The day after prolonged sitting or standing the subjects completed a postprandial high fat/glucose tolerance test, during which blood and expired gas was collected immediately before and hourly for 6 h after the ingestion of the test meal. Indirect calorimetry was used to measure substrate oxidation while plasma samples were analyzed for triglyceride, glucose, and insulin concentrations. Standing resulted in a lower fasting plasma triglyceride concentration (p = 0.021) which was primarily responsible for an 11.3% reduction in total area under the curve (p = 0.022) compared to sitting. However, no difference between trials in incremental area under the curve for plasma triglycerides was detected (p>0.05). There were no differences in substrate oxidation, plasma glucose concentration, or plasma insulin concentration (all p>0.05). These data demonstrate that 12 h of standing compared to 14 h of sitting has a small effect the next day by lowering fasting plasma triglyceride concentration, and this contributed to a 11.3% reduction in postprandial plasma triglyceride total area under the curve (p = 0.022) compared to sitting.