Most surgical procedures involve structures deeper than the skin. However, the difference in surgical noxious stimulation between skin incision and laparoscopic trocar insertion is unknown. By analyzing instantaneous heart rate (IHR) calculated from the electrocardiogram, in particular the transient bradycardia in response to surgical stimuli, this study investigates surgical noxious stimuli arising from skin incision and laparoscopic trocar insertion, and their difference. Thirty-five patients undergoing laparoscopic cholecystectomy were enrolled in this prospective observational study. Sequential surgical steps including umbilical skin incision (11 mm), umbilical trocar insertion (11 mm), xiphoid skin incision (5 mm), xiphoid trocar insertion (5 mm), subcostal skin incision (3 mm), and subcostal trocar insertion (3 mm) were investigated. IHR was derived from electrocardiography and calculated by the modern time-varying power spectrum. Similar to the classical heart rate variability analysis, the time-varying low frequency power (tvLF), time-varying high frequency power (tvHF), and tvLF-to-tvHF ratio (tvLHR) were calculated. Prediction probability (P_K ) analysis and global pointwise F-test were used to compare the statistical performance between indices and the heart rate readings from the patient monitor. Analysis of IHR showed that surgical stimulus elicits a transient bradycardia, followed by the increase of heart rate. Transient bradycardia is more significant in trocar insertion than skin incision (p < 0.001 for tvHF). The IHR change quantifies differential responses to different surgical intensity. Serial P_K analysis demonstrates de-sensitization in skin incision, but not in laparoscopic trocar insertion. Quantitative indices present the transient bradycardia introduced by noxious stimulation. The results indicate different effects between skin incision and trocar insertion.