Sensors able to record large bowel physiology and biochemistry in situ in awake rodents are lacking. Microdialysis is a mini-invasive technique that may be utilized to continuously deliver or recover low-molecular substances from various tissues. In this experiment we evaluated the feasibility of in vivo microdialysis to monitor extracellular fluid chemistry in the descending colon submucosa of conscious, freely moving rodents. Following surgical implantation of a microdialysis probe, male Wistar rats were housed in metabolic cages where they were analgized and clinically followed for four days with free access to standard diet and water. To assess local microcirculation and probe function, glucose, lactate, glucose-to-lactate ratio and urea clearance were determined in the dialysates from the three postoperative days with focus on the final 24-h period. In an attempt to mitigate the expected tissue inflammatory response, one group of animals had the catheters perfused with 5-aminosalicylic acid-enriched medium with final concentration 1 μmol/L. For verification of probe position and the assessment of the surrounding foreign body reaction, standard histological and immunohistochemical methods were employed. Microdialysis of rat gut is associated with considerable technical challenges that may lead to the loss of probe function and high drop-out rate. In this setting, limited data did not allow to draw any firm conclusion regarding local anti-inflammatory effectiveness of 5-aminosalicylic acid perfusion. Although intestinal microdialysis may be suitable for larger anesthetized animals, low reproducibility of the presented method compromises its routine experimental use in awake and freely moving small-sized rodents.