Male Tree Swallows (Tachycineta bicolor) face intense sperm competition because mated pairs copulate frequently, extra-pair copulations are common, and females store sperm. We examined the effects of repeated sampling on the characteristics of Tree Swallow semen by manually expressing semen from 15 males immediately after capture (T₀) and then hourly for 4 h (T₁–T₄). The semen characteristics of individual males varied in response to repeated sampling. The total number of sperm cells we obtained from each male over the 4-h sampling period varied from 10⁴–10⁷. Semen samples lacking sperm increased from 6.7% of T₀ samples to 26.7– 33.3% of subsequent samples. Forty percent of males provided at least one semen sample that lacked sperm. There were no significant differences among hourly samples in semen volume, sperm concentration, or in the total number of sperm cells obtained from each male. However, there were significant differences among males in each of these variables. Semen volumes represented small proportions of cloacal protuberance volumes. We did not detect significant correlations between total semen volumes or total number of sperm cells obtained from males from T₀–T₄ and cloacal protuberance volumes. Total semen volume and number of sperm cells obtained from T₀–T₄ significantly increased with date. However, sperm concentration was not significantly correlated with date. We did not detect significant correlations between semen characteristics and male morphology. Individual variation in responses to repeated sampling has implications for the copulatory strategies of male and female Tree Swallows.