And once again, there are zero American men in the US Open's round of 16 -- something that had never happened until it did last year at the country's tennis championship, which was first played in 1881.
On a windy, cloudy evening, the 13th-seeded Isner hit 42 aces, saved all five break points he faced -- and yet it wasn't enough. Unable to capitalize on plenty of openings, and surprisingly outplayed in a trio of tiebreakers, Isner lost to the 22nd-seeded Kohlschreiber 7-6 (4), 4-6, 7-6 (2), 7-6 (4).
"It's disappointing for me personally -- not for America as a whole,'' Isner said.
It was the third straight year that these two men faced each other in the third round in New York, and Kohlschreiber won them all. He eliminated the big-serving, 6-foot-10 Isner in five sets in 2012, and in four sets in 2013.
Isner only converted 1 of 12 break points he accumulated.
"Got a little tight, to be honest, and didn't move my feet on some points that I really needed," Isner said, resting his chin on his left fist. "I had chances. I just didn't convert."
The 29-year-old Isner is best known for winning the longest match in tennis history, an 11-plus-hour marathon spread over three days that ended 70-68 in the fifth set at Wimbledon in 2010.
Currently, he is the only U.S. man ranked inside the top 45, and has made clear he does not necessarily enjoy that distinction.
Fans at Louis Armstrong Stadium tried to boost their guy with chants of "Let's go, John!" And they roared throughout the fourth set, especially when Isner would edge ahead within a game.
But this one mainly came down to the tiebreakers, usually a strong suit for Isner: He entered the day 37-17 in those set-deciders this season, while Kohlschreiber was only 9-11.
"He was just better," Isner said. "I've got to be better. I know I can be. Just not showing it."
Earlier Saturday, 57th-ranked Sam Querrey -- entering the day, the only other man from the host country remaining of the 12 originally in the draw -- put up little resistance while bowing out against No. 1 Novak Djokovic 6-3, 6-2, 6-2.
Kohlschreiber now will face seven-time major champion Djokovic in the fourth round.
Other fourth-round matchups: No. 3 Stan Wawrinka, the Australian Open champion, against No. 16 Tommy Robredo, who beat 19-year-old Nick Kyrgios 3-6, 6-3, 7-6 (4), 6-3 in a match that ended after 12:30 a.m. Sunday; 2012 U.S. Open champion Andy Murray against No. 9 Jo-Wilfried Tsonga; No. 5 Milos Raonic against No. 10 Kei Nishikori. Wawrinka advanced when Blaz Kavcic withdrew because of pain in his right foot.
Given Isner's history against Kohlschreiber at the US Open, it's tough to call that result surprising, even if the American is ranked higher and is 4-0 against the German everywhere else they've played.
The Associated Press contributed to this report.