Introduction Anorectal manometry is themost well established and commonly used technique for investigating anorectal function but despite its widespread use, it has well established limitations. Functional Lumen Imaging Probe (FLIP) is a novel technique for measuring anorectal function. Its repeatability and validity of in anorectal studies has already been established. This study aimed at establishing dynamic and non-homogenous properties of anal canal with and without rectal distension, in particular to demonstrate the sampling reflex, the poorly understood physiological mechanism of which remains uncertain. Methods 19 healthy volunteers were recruited (9 females), mean age 34 (20-75). Purpose built catheters incorporating rectal and anal canal balloon were used. Appropriate sized catheter anal canal balloon (2, 3 and 4cm long) corresponding to the length of subject's anal canal (based on manometry) was used. 3 cross sectional area (CSA) readings were obtained with 2cm balloon, 5 with 3cm and 10 with 4cm balloon. In order to obtain meaningful results, the anal canal balloon was required to be touching the lumen wall. This was achieved by varying inflation volumes according to the balloon size. Rectoanal inhibition reflex (RAIR) was recorded by inflating the rectal balloon while recording the anal canal CSAs with the anal canal balloon. Participants underwent standard water-perfused anal manometry followed by FLIP on the same day. The anal canal was divided into distal, mid and proximal parts based on anatomy and preliminary data analysis. Paired t-test was done comparing CSA in the following segments (across all the balloon volumes), distal with mid anal canal, mid with proximal anal canal and distal with upper anal canal. The parameters looked at included CSA at rest, squeeze and during RAIR. Results Statistically significant difference was noted in between CSAs of the three segments both at rest and squeeze (Table 1). In all the three phases of rest, squeeze and RAIR, distal segment had the lowest mean CSA followed by mid and proximal segment respectively (Table 2). Analysis of RAIR revealed a significant difference in CSAs between proximal and distal segments (p = <.0001) but not between mid and the proximal segments (p = .351). Conclusions EndoFLIP allows detailed segmental description of the anal canal. CSA differences in three segments of anal canal clearly reveal its dynamic nature. The distal-most part of anal canal has the lowest distension followed by mid and proximal part, both at rest and squeeze. This segmental difference in the anal profile demonstrates the anatomical and physiological basis of the sampling reflex. Segmental cross sectional areas