Lleyton Hewitt, the 2002 Wimbledon winner, has been knocked out of the 2013 Championships by flamboyant German qualifier Dustin Brown.
The 32-year-old was beaten 6-4 6-4 6-7 (3-7) 6-2 after struggling to handle 6ft 5in Brown's unorthodox style.
Hewitt briefly threatened a comeback, winning the third set, but Brown triumphed in two hours and 26 minutes.
Jo-Wilfried Tsonga is also out after withdrawing with a knee injury while trailing Ernests Gulbis 3-6 6-3 6-3.
The number six seed, who could have been Andy Murray's quarter-final opponent, needed a medical timeout at the end of the second set but continued to struggle with his movement.
He was the seventh withdrawal of an injury-ridden day at SW19.
Brown, will now face Adrian Mannarino of France who went through courtesy of Isner's retirement.
"It's going to take a while to sink in," said the world number 189. "I don't normally cry, I'm playing Lleyton Hewitt, a guy I watched growing up, and it's emotional.
"I've been so often close to winning top matches and I pulled it together for this one. It was tough to stay focused and I just tried to stay in it. The way I was playing and serving, people have to beat me the way I want to play, not the way they do."
Julien Benneteau, seeded 31, is out after he was beaten 7-6 (7-1) 7-6 (7-4) 6-4 by Spaniard Fernando Verdasco while number 20 seed Mikhail Youzhny was taken to five sets by Canadian Vasek Pospisil.
He eventually secured his place in round three with a 6-2 6-7 (7-3) 7-6 (9-7) 3-6 6-4 win.