Pigs are widely used in metabolic research with procedures often requiring general anaesthesia. The aim was to investigate the effect of four different anaesthetic protocols: 1) isoflurane inhalation, 2) propofol infusion, 3) a mixture of tiletamine, zolazepam, medetomidine, ketamine and butorphanol (TZMKB)) and 4) ketamine combined with midazolam and xylazine (KMX)) on selected biomarkers during basal and glucose stimulated conditions. Eight domestic pigs were included in a cross-over design. Plasma concentrations of glucose, insulin, C-peptide, glucagon, cortisol, triglycerides, total cholesterol, aspartate amino transferase and alanine amino transferase, creatinine, urea, fructosamine, albumin, free fatty acids (FFAs) and glycerol were measured at baseline, during 2 h of anaesthesia and during 1 h of recovery. Intravenous glucose tolerance test (IVGTT, 0.5 g glucose/kg) was performed after 1 h of anaesthesia. Glucose disappearance rate and areas under the insulin, C-peptide and glucagon curves from the IVGTT were calculated. All four anaesthetic protocols affected glucose metabolism parameters significantly compared with un-anaesthetised pigs, which was particularly evident during IVGTT and for TZMKB and KMX anaesthesia. Propofol additionally influenced the plasma concentrations of triglycerides, FFAs and glycerol significantly. The remaining circulating biomarkers were largely unaffected by anaesthesia. These data underline the importance of considering the anaesthetic protocol in porcine studies of circulating metabolic biomarkers.