Physical inactivity is considered to be deleterious to vascular health, and in particular in first-degree relatives to patients with type 2 diabetes (FDR) and persons born with low birth weight (LBW), who may later in life develop cardiovascular disease. A period of imposed physical inactivity could unmask this risk. We hypothesized that the impact of physical inactivity on endothelial function would be more marked in subjects at increased risk for type 2 diabetes and cardiovascular disease (LBW and FDR) compared with a matched control group (CON), all of whom were recruited via advertisements and via the Danish Birth Registry. Twenty LBW, 20 CON and 13 FDR were studied before and after 10 days of bed rest. Forearm blood flow (FBF) was measured by venous occlusion plethysmography during brachial intra-arterial infusion of acetylcholine or adenosine at baseline and with superimposed hyperinsulinaemia. Markers of endothelial activation and inflammation were measured in plasma. Bed rest did not change the vasodilator responses to adenosine or acetylcholine alone in any group, but reduced vasodilator responses to adenosine or acetylcholine during hyperinsulinaemia in LBW. Bed rest impaired insulin-mediated vasodilatation in CON and LBW and increased endothelial activation markers in FDR and LBW but not in CON. Vasodilator responses were very low in FDR prior to bed rest, and did not decrease further during bed rest. Physical inactivity does not impair endothelium-dependent vasodilatation per se, but the vascular vasodilator effect of insulin diminished in CON and LBW after bed rest. In FDR, a further deterioration of FBF with inactivity is not possible.