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Introduction: Although NOTES may offer decreased postoperative pain, wound complications, and improved cosmesis compared to standard laparoscopic surgery, its immunologic impact is unknown. NOTES requires the creation and closure of a viscotomy, potentially contaminating the normally sterile peritoneal cavity. We hypothesize that NOTES cholecystectomy is associated with increased systemic cytokine activation compared to laparoscopic cholecystectomy (LAP).Methods: Patients requiring cholecystectomy were recruited. Exclusion criteria were age > 65 yrs, body mass index (BMI) >40 kg/m2, choledocholithiasis, acute cholecystitis, prior pelvic or gastric surgery, endometriosis, steroid use, and immunosuppression. NOTES patients underwent hybrid transvaginal or transgastric cholecystectomy. LAP patients underwent standard laparoscopic cholecystectomy. Laparoscopic roux-en-y gastric bypass (RYGB) patients were enrolled as a positive control group. Peripheral venous blood was collected preoperatively (Baseline) and 30 minutes postoperatively (Postop). Plasma cytokine analysis was done using multiplex ELISA arrays for IL-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-15, IL-17, IL-23, IFN-gamma, TNF-alpha, and TNF-beta. IL-6 and IL-10 were chosen as primary end-points based on a priori power analysis using preliminary data, with additional cytokines treated as secondary end-points. Cytokine levels were analyzed using a mixed-effects model. Results: 21 subjects underwent LAP (n=8), NOTES (n=5), or RYGB (n=8). Mean age ± SD (37 ± 12 v. 42 ± 11 yrs), BMI (29 ± 5 v. 30 ± 4 kg/m2), and male sex (n=2 v. n=2) were similar between LAP and NOTES groups, respectively. Postop IL-6 was higher for NOTES versus LAP (69 ± 35 v. 22 ± 24 pg/ml, p < 0.05). IL-10 was higher for RYGB versus LAP (56 ± 36 v. 14 ± 14 pg/ml, p < 0.05). No significant differences were seen in levels of the secondary end-points.Conclusion: NOTES cholecystectomy is associated with increased early postoperative activation of IL-6, and possibly IL-10 compared to LAP. NOTES may cause a greater physiologic insult compared to laparoscopic surgery.