Prolonged, uninterrupted sitting is demonstrated to acutely impair glucose homeostasis, but it also leads to detrimental cardiovascular health effects. We examined whether ischemic preconditioning (IPC) prevents the impact of prolonged sitting-induced glucose intolerance and measured related influencing factors such as (para)sympathetic nerve activity [assessed by heart rate variability (HRV)] and blood pressure during 2 h of prolonged sitting. In this randomized, controlled crossover study, 15 healthy participants (80% men) with a mean age of 21 +/- 1 yr (means +/- SD) and body mass index of 25.0 +/- 2.4 kg/m(2) performed IPC (IPC, 4 x 5-min 220-mmHg unilateral occlusion at the thigh muscle) or a sham intervention (sham, 4 x 5 min 20-mmHg), followed by 2 h of sitting. After IPC or sham intervention, fingertip blood glucose was measured before and after 30, 60, 90, and 120 min of 75 g of glucose ingestions. Blood glucose responses during an oral glucose tolerance test were significantly attenuated, resulting in a lower area under the curve when sitting was preceded by a bout of IPC than sham (P < 0.05). IPC increased high-frequency oscillations and decreased the ratio of low-frequency-to-high-frequency oscillations at 120 min in HRV (P < 0.05). Moreover, a lower blood pressure was observed with IPC compared with sham (P < 0.05). Prolonged sitting or IPC did not affect cerebrovascular responses (P > 0.05). Collectively, these results indicate that the application of IPC before prolonged, uninterrupted sitting bout was associated with a better glucose tolerance and prevented impairment in (para)sympathetic nerve activity and blood pressure in healthy young men and women.