What is the central question of this study? To understand better the effects of acute hyperglycaemia on arterial stiffness in healthy young individuals, we assessed arterial stiffness in physically active men before and after reduced ambulatory physical activity to decrease insulin sensitivity. What is the main finding and its importance? During an oral glucose tolerance test, we identified an increase in leg arterial stiffness (i.e. reduced femoral artery compliance) only when subjects were inactive for 5days (<5000stepsday-1 ) and not when they were engaging in regular physical activity (>10,000stepsday-1 ). These results demonstrate the deleterious consequence of acute reductions in daily physical activity on the response of the peripheral vasculature to acute hyperglycaemia. Acute hyperglycaemia has been shown to augment indices of arterial stiffness in patients with insulin resistance and other co-morbidities; however, conflicting results exist in healthy young individuals. We examined whether acute hyperglycaemia after an oral glucose tolerance test (OGTT) increases arterial stiffness in healthy active men before and after reduced ambulatory physical activity to decrease insulin sensitivity. High-resolution arterial diameter traces acquired from Doppler ultrasound allowed an arterial blood pressure (BP) waveform to be obtained from the diameter trace within a cardiac cycle. In 24 subjects, this method demonstrated sufficient agreement with the traditional approach for assessing arterial compliance using applanation tonometry. In 10men, continuous recordings of femoral and brachial artery diameter and beat-to-beat BP (Finometer) were acquired at rest, 60 and 120min of an OGTT before and after 5days of reduced activity (from >10,000 to<5000stepsday-1 ). Compliance and β-stiffness were quantified. Before the reduction in activity, the OGTT had no effect on arterial compliance or β-stiffness. However, after the reduction in activity, femoral compliance was decreased (rest, 0.10±0.03mm2 mmHg-1 versus 120min OGTT, 0.06±0.02mm2 mmHg-1 ; P<0.001) and femoral β-stiffness increased (rest, 8.7±2.7a.u. versus 120min OGTT, 15.3±6.5a.u.; P<0.001) during OGTT, whereas no changes occurred in brachial artery compliance (P=0.182) or stiffness (P=0.892). Insulin sensitivity (Matsuda index) was decreased after the reduction in activity (P=0.002). In summary, in young healthy men the femoral artery becomes susceptible to acute hyperglycaemia after 5days of reduced activity and the resultant decrease in insulin sensitivity, highlighting the strong influence of daily physical activity levels on vascular physiology.