Third seeded Spaniard Muguruza, who has struggled in the aftermath of her Roland Garros breakthrough in June, slumped to a 7-5, 6-4 loss to Latvia’s Anastasija Sevastova, the world number 48.
A horribly out of sorts Muguruza rallied from 1-5 down in the second set, saving three match points, but Sevastova, who briefly quit the sport in 2013, held her nerve for the victory.
Having not won a match at the US Open in five years before this week, 26-year-old Sevastova now goes on to face Kateryna Bondarenko, a 5-7, 7-6 (7/5), 7-5 winner in three hours over China’s Zheng Saisai.
“This is why I came back to the sport for the big stages like this and night sessions at the Slams. But I was shaking a little at the end,” she said.
Muguruza admitted she had been outplayed.
“It was a very tough match. I think I didn’t play well. Everything she was doing was working,” said the 22-year-old who has never made the third round in New York.
Meanwhile, 120th-ranked Ryan Harrison of the United States stunned fifth seeded Raonic 6-7 (4/7), 7-5, 7-5, 6-1 in another second round upset.
“It was extremely physical out there. I was playing on adrenaline,” said 24-year-old qualifier Harrison who was as high as 43 in the world in 2012.
He goes on to face experienced Cypriot Marcos Baghdatis for a place in the last 16.
Harrison has now reached the third round of a Slam for the first time after recording just a second win over a top 10 opponent in 27 meetings.
Raonic, considered as a potential title dangerman in New York, hit 69 winners but committed 62 unforced errors.
“I started cramping midway through the second set. There was a lot of nerves and stress. It was a result of over-exuberance,” said 25-year-old Raonic.
– Nadal first winner under roof –
Rafael Nadal, the 2010 and 2013 champion, eased past Italy’s Andreas Seppi 6-0, 7-5, 6-1 in a match which became the first to be completed under the new retractable roof on Ashe.
Light rain caused the structure to be closed at 3-3 in the second set with play only held up for around seven minutes.
“It’s part of history and I am very happy to be the first to play under the roof,” said Nadal, who was also the first to practice beneath the roof on the cavernous 22,000-capacity arena.
Nadal beat Seppi for the eighth time in nine matches and next faces Russia’s Andrey Kuznetsov.
World number one Novak Djokovic made the last 32 of a major for the 33rd successive time without hitting a ball when Jiri Vesely of the Czech Republic withdrew from their second round encounter suffering an inflammation of the left forearm.
Djokovic will face 34-year-old Russian Mikhail Youzhny, twice a semi-finalist, on Friday for a place in the last 16.
– Konta drama –
Britain’s Johanna Konta, the 13th seed, collapsed on court with heat sickness before completing a 6-2, 5-7, 6-2 win over Bulgaria’s Tsvetana Pironkova.
After a medical timeout, the Briton dropped the second set before heading off on a lengthy toilet break which did little to help her opponent’s composure in the decider.
“I’m a little embarrassed. I’m not a fan of drama. I did the best that I could with the cards that I had,” said Konta.
Angelique Kerber, the second seeded Australian Open champion, made the last 32 by seeing off 34-year-old Mirjana Lucic-Baroni of Croatia 6-2, 7-6 (9/7).
Kerber faces 17-year-old CiCi Bellis of the United States, for a place in the last 16.
Croatian seventh seed Marin Cilic, the 2014 champion, made the last 32 by seeing off Sergiy Stakhovsky 6-1, 6-2, 6-3.