Background There is no standardization of the use of suction during puncturing of a target in pancreatic EUS-guided FNA (EUS-FNA). It is also debatable whether expressing aspirates from the needle by the traditional method of reinserting the stylet is more effective than by air flushing, which is easier and safer. Objective To optimize sampling techniques in pancreatic EUS-FNA. Design Prospective, comparative trial. Setting Tertiary-care referral center. Patients Eighty-one consecutive patients with solid pancreatic masses. Intervention Four punctures were performed for each mass in random order by a 2 × 2 factorial design. Sample quality and diagnostic yield were compared between samples with suction (S+) versus no suction (S-) and expressed by reinserting the stylet (RS) versus air flushing (AF). Main Outcome Measurements Sample quality by the number of diagnostic samples, cellularity, bloodiness, and air-drying artifact; diagnostic yield by accuracy, sensitivity, and specificity. Results The number of diagnostic samples (72.8% vs 58.6%; P = .001), cellularity (odds ratio OR 2.12; 95% confidence interval CI, 1.37-3.30; P < .001), bloodiness (OR 1.46; CI, 1.28-1.68; P < .001), accuracy (85.2% vs 75.9%; P = .004), and sensitivity (82.4% vs 72.1%; P = .005) were higher in S+ than in S-. Bloodiness was lower in AF than in RS (OR 1.16; CI, 1.03-1.30; P = .017). Limitations Single-center trial, 2 kinds of needle gauges, and no immediate cytopathology evaluation. Conclusion Puncturing with suction and expressing by air flushing may be used preferentially in pancreatic EUS-FNA because they were more effective and convenient techniques. (Clinical trial registration number: NCT01354795 .)