Background: It has been shown, in animal models, that the gastrointestinal tract (GIT) motility is influenced by temperature; nevertheless the basic mechanism governing thermal GIT smooth muscle responses has not been fully investigated. Studies based on physiologically tuned mathematical models predict that thermal inhomogeneity may induce an electrochemical destabilization of the peristaltic activity. Herewith, the effect of thermal cooling on human colonic muscle strip (HCMS) contractility was studied. Methods: HCMSs were obtained from disease-free margins of resected segments for cancer. After removing the mucosa and serosa layers, strips were mounted in separate chambers. After 30 minutes, spontaneous contractions developed, which were measured using force displacement transducers. Temperature was changed every hour (37, 34, 31°C). The effect of cooling was analyzed on mean contractile activity, oscillations amplitude, frequency and on contraction to Acetylcholine (Ach, 10^-5M). Key Results: At 37°C HCMSs developed a stable phasic contraction (~ 0.02 Hz) with a significant Ach-elicited mean contractile response (31%, 22% compared to baseline in circular and longitudinal axis, respectively). Lowering bath temperature, higher mean contractile amplitude was observed and it increased in presence of Ach (78% and 43% higher than the basal tone in the circular and longitudinal axis, respectively, at 31 °C). A simplified thermo-chemo-mechanical model is tuned on experimental data characterizing the stress state coupling the intracellular calcium concentration to tissue temperature. Conclusions: Acute thermal cooling affects colonic muscular function. Further studies are needed to establish the mechanisms involved in order to better understand clinical consequences of hypothermia on intestinal contractile activity.