Gavage feeding is required in preterm infants who cannot feed by themselves. Insertion of the feeding tube is painful, and reducing the discomfort in these patients is desirable. The aim of this study was to assess pain and discomfort during nasal insertion of a feeding tube, and to evaluate different measures for pain relief. We included 24 preterm infants with postmenstrual age 28 to 32 weeks' who were in stable condition. Each infant acted as his or her own control over a 3-week period during which the tube was changed 6 times. On these occasions, 6 different treatment combinations were given in randomized order: pacifier or no pacifier, combined with no fluid, sterile water, or 30% sucrose. Pain and discomfort were assessed by at least 2 independent and experienced observers using a pain assessment tool, the Premature Infant Pain Profile; score range: 0 to 21. In general, scores of 4 to 6 are interpreted as normal or no discomfort; ≥ 12 usually signals significant pain and distress. The median Premature Infant Pain Profile score during the procedure was 9 and decreased gradually toward 4 after 5 minutes. The lowest pain score was achieved by combining a pacifier with oral sucrose. Sterile water without a pacifier gave the highest score. Insertion of a feeding tube in preterm infants leads to a measurable degree of pain and discomfort, according to the Premature Infant Pain Profile assessment tool. Pain relief was best achieved by combining a pacifier with 30% sucrose.