Background: The treatment of complex anal fistulae remains a therapeutic challenge. Early anal fistula plug (AFP) studies showed high success rates, while later reports demonstrated success rate of 13-100% at usual follow-up of 6-24 months. Very little is known about morphological findings related to success and failure of the AFP, especially if three-dimensional (3D) endoanal ultrasound (EAUS) is used for radiological follow-up. No documentation is available for the use of a high-resolution transducer for assessment of anal fistulae and anal canal length.Aims: I: To evaluate the long-term success rate of the AFP in the treatment of complex anal fistulae. II: To explore the utilisation of 3D EAUS for the follow-up of the AFP. III: To evaluate the accuracy of 3D EAUS alone for assessment of newly diagnosed cryptoglandular fistulae, compared to intraoperative findings in combination with intraoperative 3D EAUS (gold standard). IV: To explore the use of 3D EAUS for the measurement of anal canal length in patients with newly diagnosed cryptoglandular fistulae.Methods: Two prospective cohort study projects, each designed to produce two papers. Papers I & II: A single-centre study of consecutive patients with complex anal fistulae treated in May 2006-October 2009. Patients with ano/rectovaginal fistulas were excluded. Papers III & IV: A single-centre study of consecutive patients with newly diagnosed cryptoglandular anal fistulae and no history of fistula surgery treated between June 2018-March 2020. Patients with inflammatory bowel disease, ano/rectovaginal fistulae, more than one fistula, or a fistula with more than one internal orifice (IO) were excluded.Main outcomes: Paper I: AFP success rate >7 years postoperatively, Paper II: Morphological 3D EAUS findings and relation to AFP failure, Paper III: Agreement between 3D EAUS alone and the gold standard, Paper IV: the mean anal canal length in female and male with transducers 2052 and 8838.Results and conclusions: Paper I: The overall success rate 93-138 months after the AFP was 38%. It is acceptable considering the low morbidity of the procedure in a complex disease with high recurrence rates over time.Paper II: 3D EAUS may be utilised for AFP follow-up. Postoperative 3D EAUS at three months or later, especially in combination with clinical symptoms, can be used to predict long-term AFP failure. Paper III: 3D EAUS alone has high precision in identifying IO of anal fistulae. The method may be utilised even by relatively inexperienced surgeons to identify fistulae suitable for fistulotomy.Paper IV: Measurements of anal canal length in female and male patients assessed by two different transducers for 3D EAUS are reported with generally acceptable reproducibility and repeatability.