Endothelial dysfunction result from inflammation and excessive production of reactive oxygen species as part of the surgical stress response. Remote ischemic preconditioning (RIPC) potentially exerts anti-oxidative and anti-inflammatory properties, which might stabilise the endothelial function after non-cardiac surgery. This was a single centre randomised clinical trial including 60 patients undergoing sub-acute laparoscopic cholecystectomy due to acute cholecystitis. Patients were randomised to RIPC or control. The RIPC procedure consisted of four cycles of five minutes of ischaemia and reperfusion of one upper extremity. Endothelial function was assessed as the reactive hyperaemia index (RHI) and circulating biomarkers of nitric oxide (NO) bioavailability (L-arginine, asymmetric dimethylarginine (ADMA), L-arginine/ADMA ratio, tetra- and dihydrobiopterin (BH 4 and BH 2), and total plasma biopterin) preoperative, 2-4 h after surgery and 24 h after surgery. RHI did not differ between the groups ( p = 0.07). Neither did levels of circulating biomarkers of NO bioavailability change in response to RIPC. L-arginine and L-arginine/ADMA ratio was suppressed preoperatively and increased 24 h after surgery ( p < 0.001). The BH 4/BH 2-ratio had a high preoperative level, decreased 2-4 h after surgery and remained low 24 h after surgery ( p = 0.01). RIPC did not influence endothelial function or markers of NO bioavailability until 24 h after sub-acute laparoscopic cholecystectomy. In response to surgery, markers of NO bioavailability increased, and oxidative stress decreased. These findings support that a minimally invasive removal of the inflamed gallbladder countereffects reduced markers of NO bioavailability and increased oxidative stress caused by acute cholecystitis.