Tenth seed Victoria Azarenka suffered a shock straight-set defeat by world number 161 Anna Karolina Schmiedlova in the French Open second round.
The Belarusian, 31, reached the US Open final earlier this month but struggled on the clay against Slovak Schmiedlova, who won 6-2 6-2.
American teenage sensation Coco Gauff also lost, struggling with serve in a 4-6 6-2 7-5 defeat by Martina Trevisan.
Top seed Simona Halep and third seed Elina Svitolina both won their matches.
Halep, who won her first Grand Slam title at the 2018 Roland Garros, beat fellow Romanian Irina Camelia Begu 6-3 6-4.
- Follow day four of the French Open
- Serena Williams withdraws from Roland Garros
- Bertens leaves court in a wheelchair after bad-tempered win
Fresh from winning the Italian Open title earlier this week, 29-year-old Halep demonstrated why she is considered the favourite in Paris with a clean performance to earn her 15th straight win.
Halep will face 25th seed Amanda Anisimova in the third round, giving her the chance to avenge defeat by the American teenager in last year's quarter-finals.
Anisimova, 19, teed up the rematch with a 6-2 6-0 win over fellow American Bernarda Pera.
Ukraine's Svitolina regained her composure after being bagelled in the second set to claim a 6-3 0-6 6-2 victory over Mexican qualifier Renata Zarazua.
"I was trying to get back into the match," said the 26-year-old. "I lost my way a bit and was making too many errors."
She was also briefly taken aback by a sonic boom caused by a French fighter jet, saying: "I was a bit worried because I thought something bad happened."
The military said the plane was scrambled to aid two commercial aircraft that had lost radio contact and was authorised to travel at supersonic speed.
Svitolina, who won her 15th WTA title in Strasbourg last week but is yet to reach a Grand Slam final, will face Russian world number 31 Ekaterina Alexandrova next.
A possible meeting with Serena Williams in the quarter-finals is no longer on the cards after the American 23-time Grand Slam champion withdrew before her second-round match with an Achilles injury.
World number 51 Gauff, 16, ended her second-round match with a total of 19 double faults to gift victory to Italian Trevisan, who is ranked 159th in the world.
The service errors were ultimately the American's undoing as two in the final game gave Trevisan match point and Gauff's mis-hit forehand landed wide to bring her first French Open main-draw run to a close.
Gauff has grabbed headlines in the past 15 months, having reached the fourth round at Wimbledon and the Australian Open, but this defeat comes soon after a first-round exit at the US Open.
- How Do You Cope?: Fabrice Muamba on the night that changed his life
- Football Daily: All the reaction from some heavyweight EFL Cup ties