The 6-foot-10 American now stands alone with 13,748 untouchable serves and counting, passing Ivo Karlovic's previous leading mark of 13,728.
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Isner needed just two service games to break the record Friday on Wimbledon's No. 2 Court, firing the record-breaking ace at 1-1, 15/30. He finished the match with 24 aces in a 4-6, 6-7(4), 3-6 defeat.
"I'm actually very proud of that record," Isner reflected post-match. "I wish I could have broken it in a win, but to have set the all-time aces record at Wimbledon I think is very special. It's a credit to — in my mind — how professional I've been to keep myself healthy for such a long time, to keep myself on the court to give myself the opportunity to hit those aces.
"It is something I'm actually pretty proud of. I don't pat myself on the back too much, but this one I think is pretty cool."
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The American also leads the 2022 ace race with 653 on the season following Friday's match. The 37-year-old has finished first on the ATP Tour in aces in seven seasons and eclipsed the 1,000-aces mark seven times, including a career-high of 1,260 in 2015. Isner accomplished both feats for the first time in 2010 after turning pro in 2007 following a standout career at the University of Georgia.