Background New hemostatic technologies are often employed in open and laparoscopic surgery to reduce duration of surgery and complications. Monopolar diathermy, Harmonic scalpel and LigaSure are routinely used in open and laparoscopic surgery for tissue cutting and hemostasis. We compared lateral thermal damage following in vivo application of 3 commonly used instruments. Methods We used monopolar diathermy, Harmonic scalpel and LigaSure to coagulate and divide the peritoneum of patients who underwent median laparotomy. After anesthesia, median supraumbilical laparotomy was performed, and the peritoneum of each patient was coagulated using different devices. Using light microscopy and morphometric imaging analysis, the width of tissue lateral thermal damage was measured from the point of the peritoneal incision. Results We included 100 patients in our study. After a peritoneal incision, the mean lateral thermal damage of monopolar diathermy, Harmonic scalpel (output power 3), Harmonic scalpel (output power 5) and LigaSure were 215.79 μm, 90.42 μm, 127.48 μm and 144.18 μm, respectively. Conclusion The degree of lateral thermal spread varied by instrument type, power setting and application time. LigaSure and Harmonic scalpel were the safest and most efficient methods of tissue coagulation. Monopolar diathermy resulted in the greatest degree of thermal damage in tissues.