In the United States, it is recommended that healthcare providers offer influenza vaccination by October, if possible. However, if the vaccine’s effectiveness soon begins to wane, the optimal time for vaccination may be somewhat later. We examined whether the effectiveness of influenza vaccine wanes during the influenza season with increasing time since vaccination. We identified persons who were vaccinated with inactivated influenza vaccine from 1 September 2010 to 31 March 2017 and who were subsequently tested for influenza and respiratory syncytial virus (RSV) by a polymerase chain reaction test. Test-confirmed influenza was the primary outcome and days-since-vaccination was the predictor of interest in conditional logistic regression. Models were adjusted for age and conditioned on calendar day and geographic area. RSV was used as a negative-control outcome. Compared with persons vaccinated 14 to 41 days prior to being tested, persons vaccinated 42 to 69 days prior to being tested had 1.32 (95% confidence interval [CI], 1.11 to 1.55) times the odds of testing positive for any influenza. The odds ratio (OR) increased linearly by approximately 16% for each additional 28 days since vaccination. The OR was 2.06 (95% CI, 1.69 to 2.51) for persons vaccinated 154 or more days prior to being tested. No evidence of waning was found for RSV. Our results suggest that effectiveness of inactivated influenza vaccine wanes during the course of a single season. These results may lead to reconsideration of the optimal timing of seasonal influenza vaccination.