Background: Hand laterality has an impact on surgical gestures. In this study, we sought to measure the multi-parameter variability of the microsurgical gesture depending on the hand used and the differences between expert microsurgeons and novices. Methods: Ten experienced microsurgeons and twenty medical students with no prior microsurgical experience performed arterial anastomosis on a chicken wing artery using dominant and non-dominant hands. We measured time and force using a homemade force-sensing microsurgical needle holder, heart rate variability with a Polar H10 chest strap, anxiety with the STAI-Y questionnaire and anastomosis quality using the MARS 10 scale. Results: In the microsurgeons' group, duration of anastomosis (p = 0.037), force applied to the needle holder (p = 0.047), anxiety (p = 0.05) and MARS10 (p = 0.291) were better with the dominant hand. For novices, there was no difference between the dominant and non-dominant hand pertaining to force, time and stress level. There were no differences between microsurgeons and novices pertaining to force and anxiety using the non-dominant hand. Conclusions: The study highlighted a marked laterality among microsurgical experts, a finding that may be explained by current learning methods. Surprisingly, no laterality is observed in students, suggesting that for a specific gesture completely different from everyday tasks, laterality is not predefined. Ambidexterity training in the residency curriculum seems relevant and may help microsurgeons improve performance and postoperative outcomes.