Andy Murray's reign as Queen's champion came to an abrupt end as the world number four suffered a shock 6-3, 6-7 (4/7), 7-6 (7/1) defeat to France's Nicolas Mahut, while four-time winner Andy Roddick also bowed out in the second round on Wednesday.
Top seed Murray had won the pre-Wimbledon grass-court event in west London twice in the last three years, beating Jo-Wilfried Tsonga in last year's final, but he paid the price for an error-strewn display as former Queen's runner-up Mahut enjoyed one of the best wins of his career.
It was the worst possible preparation for Murray ahead of Wimbledon, which starts later this month, and leaves the Scot without a title since his triumph in Brisbane in January.
"I don't think it went all wrong. It was a very close match but losing serve in the third set after breaking him wasn't great. It got him back into the match," Murray said.
"I didn't move very well at the start of the match, but he's a very good grass court player. He's been in the final here and he played well today."
Murray has been struggling with a back injury for several weeks and was troubled by the problem during his recent run at the French Open, which ended with a defeat against David Ferrer in the quarter-finals.
He never looked completely at ease on court on Wednesday.
After failing to convert two set points in the opening game, Murray struggled to find any momentum and Mahut made him pay in the sixth game, securing the key break before serving out the first set.
It needed a tie-break to decide the second set and Murray took it as he produced a flurry of winners at the crucial moment.
Murray broke again in the first game of the final set when Mahut double faulted, but gifted the break straight back in the next game and a tie-break was required to settle the match.
Remarkably it was Mahut who took control, winning six straight points to leave Murray stunned and out of the tournament.
"This is maybe the best win of my career, it has made me very happy," Mahut said. "I'm very confident in my game and I can do more yet."
Roddick fared no better as France's Edouard Roger-Vasselin clinched a shock 6-4, 4-6, 7-5 victory over the seventh seed.
The American has slipped out of the top 30 in the world rankings after an injury-ravaged campaign, but losing to Roger-Vasselin, ranked 67th, on the grass he regards as his favourite surface is a new low.
It was the 29-year-old's 11th defeat in 18 matches this year and raises doubts about his ability to mount a strong challenge at Wimbledon, where he has reached the final three times.
Roddick's body is showing signs of wear and tear after over 10 gruelling years on the ATP Tour and the former world number one has struggled with ankle and hamstring injuries this year.
He looked sluggish again here. After losing the first set, Roddick hit back to get the only break of a hard-fought second set.
But the former US Open champion couldn't convert a match point at 5-4 in the final set and Roger-Vasselin made him pay as he broke for a 6-5 lead before serving out a shock win.
"I've certainly played a lot worse this year," Roddick said.
"I thought I had a chance there in the third set, and honestly I felt like I was going to take that thing once I turned it. It didn't work out that way."
Meanwhile, Taiwan's Lu Yen-Hsun won 6-7 (3/7), 7-6 (8/6), 7-6 (9/7) against Croatian 16th seed Ivo Karlovic.
Italy's Simone Bolelli also claimed an impressive scalp as he defeated French fourth seed Gilles Simon 4-6, 6-3, 7-6 (7/2).
Spain's Feliciano Lopez, the fifth seed, beat Steve Darcis 6-3, 3-6, 7-5, while sixth seed Marin Cilic demolished Australia's Matthew Ebden 6-2, 6-1.