Background: During laparoscopic surgery, CO₂ insufflation gas could leak from the intra-abdominal cavity into the operating theater. Medical staff could therefore be exposed to hazardous substances present in leaked gas. Although previous studies have shown that leakage through trocars is a contributing factor, trocar performance over longer periods remains unclear. This study investigates the influence of prolonged instrument manipulation on gas leakage through trocars. Methods: Twenty-five trocars with diameters ranging from 10 to 15 mm were included in the study. An experimental model was developed to facilitate instrument manipulation in a trocar under loading. The trocar was mounted to a custom airtight container insufflated with CO₂ to a pressure of 15 mmHg, similar to clinical practice. A linear stage was used for prolonged instrument manipulation. At the same time, a fixed load was applied radially to the trocar cannula to mimic the reaction force of the abdominal wall. Gas leakage was measured before, after, and during instrument manipulation. Results: After instrument manipulation, leakage rates per trocar varied between 0.0 and 5.58 L/min. No large differences were found between leakage rates before and after prolonged manipulation in static and dynamic measurements. However, the prolonged instrument manipulation did cause visible damage to two trocars and revealed unintended leakage pathways in others that can be related to production flaws. Conclusion: Prolonged instrument manipulation did not increase gas leakage rates through trocars, despite damage to some individual trocars. Nevertheless, gas leakage through trocars occurs and is caused by different trocar-specific mechanisms and design issues.