Objective: Patients undergoing hemodialysis are repeatedly exposed to stress and pain from approximately 300 punctures per year to their arteriovenous fistula. This study was designed to measure pain associated with venepuncture during AVF cannulation and to compare the effectiveness of ethyl chloride vapocoolant spray, topical eutectic mixture of local anesthetics (EMLA) cream and placebo in controlling pain caused by venepuncture of arteriovenous fistula patients undergoing chronic hemodialysis. Methods: This randomized, placebo-controlled, crossover study, included 41 patients undergoing conventional hemodialysis three times a week. First intervention was conducted as baseline pain assessment (control). In the three consecutive dialysis sessions, every patient randomly received 1) ethyl chloride vapocoolant spray, 2) EMLA, or 3) placebo cream before venepuncture. Pain perception was recorded by patients immediately after cannulation on a 0-100 mm visual analogue scale (VAS). p<0.05 was considered as significant. Results: VAS scores presented a marked inter-individual variation during venepuncture. EMLA application resulted in significantly lower total pain scores compared to control and all other interventions (p<0.05). No patient experienced severe pain with EMLA or vapocoolant. The patients reported less moderate and severe pain with EMLA, and vapocoolant spray compared to control and placebo interventions. Moderate and severe pain scores were similar between EMLA and vapocoolant spray (p>0.05). Conclusion: Venipuncture for AVF cannulation causes mild to moderate pain in hemodialysis patients. Although local application of EMLA is more effective than in preventing venepuncture pain, ethyl chloride vapocoolant is as effective as EMLA for preventing mild to moderate puncture pain in patients undergoing hemodialysis.