Background Successful cannulation is an important prerequisite for a functional arteriovenous fistula (AVF). Reasons for unsuccessful cannulation of an AVF are multifactorial and poorly evaluated. In our experience, a needle access segment (NAS) with a length of 10 cm, <5 mm deep from the skin surface, and >6 mm diameter assessed objectively using duplex Doppler ultrasound (DDUS) imaging, in a fistula with brachial artery flow >500 mL/min, permits consistent cannulation. This report provides observational data on the NAS of the outflow veins after fistula creation and a detailed long-term outcome on AVFs that needed superficialization of the NAS using minimal incision superficialization technique (MIST) to make them suitable for cannulation. This report is based on prospectively collected data with a longitudinal follow-up in a large patient cohort. Methods A prospective database was used to analyze consecutive patients undergoing AVF until the study end point. All patients underwent a protocol-based maturation evaluation using color DDUS imaging. Unsuitable NAS were surgically corrected using superficialization (by MIST or lipectomy) of deeply situated veins or NAS reconstruction. Results Between February 1, 2007, and May 31, 2013, 617 new AVF surgeries were performed. Outflow vein superficialization (MIST or lipectomy) or NAS reconstruction was necessary in 226 of 585 procedures (38.6%) included in this analysis. Of these, 162 (72%) were performed using MIST, 50 (22%) with a single long incision, and 14 (6%) using lipectomy technique. Technical success for MIST was 100%, and only two fistulae failed to mature. The vein depth of 9.2 ± 3.2 mm during initial vessel mapping was similar to the pre-MIST depth of 9.1 ± 3.8 mm. Depth of NAS improved to 3.1 ± 1.0 mm after MIST. The secondary patency after MIST at 6, 12, 24, 48, and 60 months was 98%, 93.3%, 88.1%, 83.3%, and 80.9%. During the 400.8 post-MIST functional fistula-years, only 0.63 procedures per year were required to maintain AVF patency. Conclusions Our data suggest that maturation of AVFs using objective criteria based on DDUS provides an opportunity to identify NAS problems in outflow veins before cannulation. Most of the of the AVF outflow veins (71.7%) could be transposed or superficialized using MIST, with excellent long-term outcomes.