BACKGROUND: Painful subcutaneous insulin injections may decrease treatment compliance. Improving injection comfort therefore represents a particular area of technological research in which steady progress has been made since the introduction of the insulin pen in 1985. Injection pain can be influenced by many variables, but relatively little is known about their impact. This study investigated the impact of injection volume (range 0-2250 µL), speed (range 0-800 µL/sec), and site (abdomen vs thigh) on pain sensation. METHOD: In random order, patients (n = 80) with type 1 or type 2 diabetes received 24 saline injections subcutaneously through a 27G ultra-thin-wall needle. Injections were performed in the abdomen (n = 19) and thigh (n = 5) with predefined speed-volume combinations. For each injected speed-volume combination, patients scored their pain sensation on a 100 mm visual analog scale (VAS). RESULTS: The mean pain scores for speed-volume combinations were all in the lower part (<20 mm) of the VAS, indicating zero to mild pain. Pain sensation was statistically higher ( P < .05) with the 2250 µL volume compared to other injection volumes (range 4.3-5.1 mm) and with thigh compared to abdomen injections (2.1 mm). Pain sensation did not change with increasing injection speed. Patient acceptance of the injection pain was high for all injections (range 93.7-98.7%). CONCLUSIONS: In summary, large volume and thigh injections are rated more painful, but the clinical impact of these findings is likely marginal considering the low absolute pain levels and high patient acceptance rates. Injection speed does not influence pain sensation.