Dominic Thiem suffered a shock loss in the Rio Open first round, while Juan Martin del Potro marked his return with a win on Tuesday.
Thiem, the top seed and 2017 champion in Rio de Janeiro, was stunned 6-3 6-3 by Serbian Laslo Djere at the ATP 500 event.
Djere, the 23-year-old world number 90, broke serve five times in an impressive performance.
Thiem was one of six seeds to fall at the rain-hit tournament.
Fabio Fognini, the Italian second seed, was comfortably beaten by Canadian wildcard Felix Auger-Aliassime 6-2 6-3.
Marco Cecchinato was edged by Aljaz Bedene 7-5 7-6 (7-1) and Dusan Lajovic was thrashed by Cameron Norrie 6-2 6-1.
The seventh-seeded Malek Jaziri went down to Federico Delbonis 6-3 7-5, while Nicolas Jarry lost to Roberto Carballes Baena 6-3 6-7 (5-7) 7-6 (8-6) in a three-hour marathon at the clay-court event.
Joao Sousa was the only seed to progress, while there were also wins for the likes of Albert Ramos-Vinolas and Christian Garin.
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At the Delray Beach Open, Del Potro made his return from a knee injury with a 6-3 7-5 win over Yoshihito Nishioka.
Playing for the first time since October, the 2011 champion sent down 12 aces to win in one hour, 51 minutes.
Unlike in Rio, it was a decent day for the seeds on the hard-courts in Florida.
John Isner got past Peter Polansky 6-3 7-6 (7-4), Steve Johnson overcame Jason Jung 7-6 (7-3) 6-4 and Andreas Seppi beat Bernard Tomic 6-4 7-5.
Adrian Mannarino was a comfortable 6-2 6-2 winner over Brayden Schnur, who was coming off a run to the New York Open final.
Reilly Opelka, who won his maiden ATP Tour title in New York, advanced with a 6-4 6-0 victory over Tennys Sandgren.
However, defending champion Frances Tiafoe went down to qualifier Dan Evans 3-6 7-6 (7-1) 7-5 and Taylor Fritz was beaten by Mackenzie McDonald 3-6 6-4 6-3.
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