The International Anorectal Physiology Working Group (IAPWG) suggests a standardized protocol to perform high-resolution anorectal manometry. The applicability and possible limitations of the IAPWG protocol in performing three-dimensional high-definition anorectal manometry (3D-ARM) have still to be extensively evaluated. The IAPWG protocol was applied in performing 3D-ARM. Anorectal manometry (ARM) and a balloon expulsion test (BET) were performed according to IAPGW protocol in 290 patients. A total of 84 males and 206 females (mean age 57.1 ± 15.7years) were enrolled in six Italian centers. The reasons for which the patients were sent to perform 3D-ARM were: constipation (53.1%), fecal incontinence (26.9%), anal pain (3.1%), postsurgical (3.8%) and presurgical evaluation (4.8%), prolapse (3.4%), anal fissure (2.8%), and other (2.1%). Due to organic and functional conditions (low rectal anterior resections, rectal prolapses, and J-pouch after colectomy), we were unable to perform a complete 3D-ARM on six patients. Overall, a complete 3D-ARM and BET following IAPWG protocol was carried out in 284 patients (97.9%). The following were recorded: rest pressure (81.9 ± 32.0mmHg) and length of the anal sphincter (37.0 ± 6.2cm), maximum anal squeeze pressure (201.6 ± 81.3mmHg), squeeze duration (22.0 ± 8.8s), maximum rectal (48.7 ± 41.0mmHg) and minimum anal pressure (73.3 ± 36.5mmHg) during push, presence/absence of a dyssynergic pattern, cough reflex and rectal sensations (first constant sensation 48.4 ± 29.5mL, desire to defecate 83.7 ± 52.1mL, and maximum tolerated volume 149.5 ± 72.6mL), and presence/absence of rectoanal inhibitory reflex. Mean 3D-ARM registration time was 14 min 7 s ± 3 min 12 s. This is the first multicentric study that evaluates the applicability of the IAPWG protocol in 3D-ARM performed in different manometric laboratories (both gastroenterological and surgical). The IAPWG protocol was easy to perform and was not time consuming. A diagnosis according to the London Classification was easily obtained in most patients in which 3D-ARM was carried out. No clear limitations to the applicability of the IAPWG protocol were detected.