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Background Near-infrared autofluorescence (NIRAF) has been introduced as an adjunct to improve parathyroid gland (PG) identification during thyroid surgery. The extent to which its intraoperative application is influenced by a learning curve remains unclear. This study aimed to evaluate and quantify a potential learning curve in NIRAF-guided thyroid surgery. Materials and methods We conducted a retrospective cohort study including all consecutive patients undergoing image-based NIRAF-guided thyroid surgery between 2021 and 2023 at two Danish ENT departments. After each procedure, all surgeons completed a standardized form detailing the manner in which NIRAF was applied. The primary outcome was the minimum number of procedures per surgeon needed to achieve consistent surgical behavior, defined as systematic use of NIRAF or a stable pattern of PG autotransplantation. Results A total of 130 patients underwent NIRAF-guided thyroid surgery, performed by two surgeons. Near-infrared autofluorescence was most often utilized on the removed specimen (94.6%), before dissection of the lower thyroid pole (93.1%) and before latero-posterior dissection (88.5%). It was less likely to be utilized before upper thyroid pole dissection (62.3%). For both surgeons, NIRAF application was not systematic in the early phase but became increasingly consistent over time (α = 11,0 (95% CI: 3,5-18,6), p=0,013 and α = 5,6 (95% CI: 0,03-11,2), p=0.049, respectively). Initially, both surgeons demonstrated remarkably high PG autotransplantation rates, which declined over time (α= -3,9 (95% CI: -7,9 - 0,07), p=0,053 and α =-4,8 (95% CI: -8,1 - (-1,6)), p=0.01, respectively). Surgeon no. 1 adopted systematic NIRAF use after a minimum of 30 procedures, while surgeon no. 2 displayed consistent behavior with regard to PG autotransplantation after a minimum of 30 procedures. Conclusions This study demonstrates a clear learning curve in NIRAF-guided thyroid surgery. A minimum of 30 procedures per surgeon was required to establish consistent surgical practice, either in terms of systematic NIRAF application or a stable pattern of PG autotransplantation. These findings underscore the importance of structured implementation strategies and targeted training when integrating the NIRAF technology into routine surgical practice.