Cameron Norrie recovered from a sloppy start to earn a first career win over Andy Murray in their all-British clash and reach the Cincinnati third round.
Norrie, 26, won 3-6 6-3 6-4 in what was only the British number one's second competitive meeting with Murray.
Murray, 35, took advantage of errors from Norrie to win the first set, but faded late in the decider as the world number 11 completed the fightback.
Norrie faces American teenager Ben Shelton, who beat Casper Ruud, next.
Shelton, 19, was given a wildcard and reached the last 16 with a 6-3 6-3 win over Norwegian fifth seed Ruud.
Australia's Nick Kyrgios lost for only the third time in 19 matches when he was beaten 6-3 6-2 by American Taylor Fritz.
Wimbledon semi-finalist Norrie and former world number one Murray were followed onto the centre court at the Western and Southern Open by British women's number one Emma Raducanu.
The 19-year-old US Open champion thrashed Belarus' former world number one Victoria Azarenka, just 18 hours after she also comprehensively beat American great Serena Williams in Tuesday's night session.
Cramps a 'big concern' for Murray
While Murray's season has again been punctuated by setbacks, notably the abdominal injury suffered in the Stuttgart final and a second-round exit at Wimbledon, it has been more encouraging than others recently.
His challenge since returning from career-saving hip surgery in 2019 has been spending more time on the court - and the world number 47 has now played more matches in 2022 than any year since 2016.
The target after losing to 20th seed John Isner at Wimbledon was improving his ranking sufficiently to be among the seeds at the upcoming US Open.
After back-to-back defeats in Washington and Montreal, a deep run was required in Cincinnati to achieve that and his already-slim hopes were ended by the defeat to Norrie.
Murray lost serve in the first game of the match before finding rhythm to win the opening set against one of his successors as Britain's leading men's player.
Norrie cut out the errors to take the second set and crucially fought off three break points, with Murray damaging a string on the first and then pushing a short forehand wide on the last, at 3-2 in the decider.
As a result, the set remained delicately poised in the latter stages until Murray began to struggle physically under consistent pressure from his younger opponent.
"At times I felt like I played really well and then at times didn't. My consistency maybe was not where I'd want it," Murray said.
"But there was some positive tennis in there at times. I definitely had enough opportunities to win that match."
Murray suffered from cramp late in a gruelling opening success against fellow veteran Stan Wawrinka on Monday and needed treatment on his upper legs after Norrie broke for a 5-4 lead.
Now starting to slow down between points, Murray showed his renowned fight, but produced some tired shots as Norrie sealed victory.
"It's a big concern for me, it's something I need to address and find a solution for," added Murray.
"No-one knows exactly why cramps happen. There are many reasons, whether it's hydration, food, fatigue, lack of conditioning, stress.
"You need to try to understand what's going on there."
Beating Andy 'massive for me' - Norrie
Norrie and Murray, who have regularly practised together and will be Davis Cup team-mates next month, shared a warm embrace at the net after the left-hander clinched victory in two hours and 37 minutes.
"It's a massive win for me, beating Andy for the first time," ninth seed Norrie said on Amazon Prime.
"I think there was not much in it. I managed to find myself at one-set all, I don't know how.
"The last set was a battle, he had some chances and I played a couple of good points."
Following his breakout Grand Slam run at Wimbledon, Norrie continued his good form by reaching the Los Cabos final, where he lost to world number one Daniil Medvedev, and the last 16 at the Rogers Cup in Montreal.
Norrie's win against Murray was his 39th on the ATP Tour this year, a tally only bettered by Spain's Carlos Alcaraz and Greece's Stefanos Tsitsipas.
"It came down to a little bit of physicality and it didn't really help him," added Norrie.
"He was struggling a little bit towards the end and I just tried to get the ball in.
"I am still struggling with the conditions a little bit and trying to find rhythm. I just got through with my attitude and stayed positive."