Berdych, the 2010 Wimbledon runner-up, broke the big-serving Isner once in each set on Sunday and won the fourth-round match 6-4, 6-4, 6-4.
The 10th-seeded Isner, who lives in Florida, had been broken only once in his first three matches at Roland Garros this year. He was the last of the eight U.S. men in the draw. No American man has made it to the quarterfinals at any Grand Slam tournament since 2011.
Isner has lost seven consecutive matches against top-10 opponents.
Berdych, a French Open semifinalist in 2010, next faces Roger Federer or Ernests Gulbis.
French Open: John Isner Goes Down to Tomas Berdych
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