Andy Murray's hopes of reaching the last 16 of a major for the first time since 2017 were crushed by Italy's Matteo Berrettini at the US Open.
Murray, 35, paid the price for a slow start as he lost 6-4 6-4 6-7 (1-7) 6-3 against the 13th seed.
Fellow Briton Jack Draper is also out after having to retire injured from his match against Russia's Karen Khachanov.
Draper, 20, levelled at one-set all, and served for the third set, but had to stop when trailing 6-5.
Murray and Draper were among the four British men to reach the third round at Flushing Meadows, setting a new record for the nation at the US Open.
British number one Cameron Norrie, seeded seventh, and 20th seed Dan Evans make up the quartet and play their last-32 matches on Saturday.
'He served well, I didn't - that was the difference'
After being hampered by a catalogue of niggles following major hip surgery in 2019, 35-year-old Murray won back-to-back matches at Flushing Meadows to reach the third round of a major for only the second time since his comeback.
That set up a tough test for the Scot, who feared he would not be able to play again after the career-threatening operation, against the powerful Berrettini.
On a bright New York afternoon, Murray struggled with his serve in the opening two sets - landing 42% of his first serves and losing three of five break points - and the constant pressure proved too much against someone of Berrettini's quality.
"I served pretty poorly for a large part of the match, which hurt me a lot. It's always tough down one end at that time of day [because of the sun]," said three-time major champion Murray.
"From one end it is understandable, but I just couldn't find any rhythm on serve."
Berrettini, who reached the 2019 semi-finals, tested Murray's movement by employing the drop shot in the first set and the tactic proved successful as the 26-year-old Italian broke for a 4-3 lead.
There had been little between the pair but a double fault on break point cost Murray dear. That gave the confidence and impetus to Berrettini to close out the set, sealing it with a hold to love completed by a unstoppable forehand winner.
A second double fault came at another inopportune time for Murray, creating a break point in the first game of the second set which Berrettini took with another forehand winner after a long rally.
Murray, now ranked 51st in the world, quickly fought back as Berrettini could not consolidate, but in the ninth game produced another double fault on a break point which allowed Berrettini to serve out for a commanding lead.
"He served extremely well, got loads of free points on his serve. I didn't. That was the difference," said Murray.
Murray pleased with physical condition in New York
If anyone could fight back from two sets down, it would be former world number one Murray.
Murray had done that on 10 previous occasions in his career, but a low first-serve percentage continued to cause him trouble at the start of the third set.
Murray had to save five break points across his opening three service games, showing more of his trademark fight to keep the set on serve and then seeing off three more break points at 5-5.
Murray's resistance was rewarded by clawing a set back with a superb tie-break, but saw what had been a tight fourth set quickly swing away from him.
After being unable to take a break point for a 4-3 lead, a poor service next game by the Briton - starting with an ill-judged drop shot for 0-15, then followed by a volley into the net and a weak backhand from the baseline - enabled Berrettini to close out victory.
That brought an end to Murray's run, 10 years on from his breakthrough major win in New York.
"I thought physically I did well, considering coming in I wasn't feeling great," said Murray, who had been suffering cramps earlier in the North American hard-court swing.
"But physically I did well in all the matches, which was good."
Draper's run cruelly cut short
At the other generational end of British men's tennis, 20-year-old Draper was aiming to further enhance his burgeoning reputation in his first appearance at this stage of a major.
Draper, playing in the main draw of an overseas Grand Slam tournament for the first time, earned an impressive win over Finland's Emil Ruusuvuori in the first round and topped that by taking the scalp of Canadian sixth seed Felix Auger-Aliassime.
His hopes of earning another notable win, this time against former world number eight Khachanov, were cruelly ruined by a hamstring injury.
"I've had problems with it before and maybe this time it was just a lot of tennis I've played in the last seven weeks maybe just caught up a little bit on me," said Draper.
"I went out to a wide ball and I just felt a twinge. I feel like when you strain something, and getting worse, there is just no going back from that really."
Draper is set to move into the world's top 50 next week after an impressive breakthrough season and again demonstrated the problems he can cause seasoned opponents.
Khachanov, seeded 27th, dominated the first set with his big serving as Draper struggled with his own first-serve percentage.
But he showed his fight and mentality to win the second set from a break down and led 5-2 in the third as 26-year-old Khachanov's mood clouded.
By that point Draper had already showed signs of the leg injury, needing a medical time-out after the fifth game, before the issue worsened.
Now struggling to push off on serve, Draper hit a double fault at break point when he served for the set at 5-4 and produced three more on his way to being broken for 6-5.
At the changeover he sat with his head in his hands and decided he had no option other than to quit after a brief chat with the physio.
Analysis
BBC tennis correspondent Russell Fuller at Flushing Meadows
That was the 16th match of Jack Draper's North American tour, which has been the longest of his life.
It was also his 56th match of the year, and for all the frustration at a hamstring problem cropping up in the third round of a Grand Slam match he looked very capable of winning, he knows his conditioning has improved dramatically over the past couple of years.
Draper was outside the world's top 250 at the start of the year: he will make his debut in the top 50 when the post-US Open list is published.
Andy Murray is moving closer to the top 40, and also thinks he has solved his recent cramping issues.
He believes it was caused by a lack of hydration, and says he has been taking a minimum of an extra half litre of sodium solution on board every hour during this US Open.