Fabio Fognini was beaten by US world number 94 Tennys Sandgren 6-3, 7-6 (12/10), 6-3 on the compact Court 14.
Novak Djokovic, winner of the last three Grand Slam tournaments, is now over 3,000 points clear of rival Rafael Nadal.
Fabio Fognini defeated Rafael Nadal twice on clay in 2015.
Novak Djokovic lost serve five times in a 6-3, 4-6, 6-2 defeat by Russia's 10th seed Daniil Medvedev.
Rafael Nadal and Roger Federer won their third round matches to move closer to a blockbuster semi-final clash at the US Open.
Tennis chiefs explained the delay in making a decision on a possible suspension on having to wait for a translation of the Fabio Fognini's brief remarks.
Defending champion Andy Murray added his voice to the growing list of unhappy players.
Gear up for the mouth watering clash as Roger Federer will again battle it out for the summit clash against Rafael Nadal on Sunday.
Rafael Nadal has never won the Miami Open despite being a four-time finalist and he expects plenty of work to get past Fabio Fognini and make it a fifth appearance.
With the two teams tied at 2-2 heading into the decisive fifth match, Fabio Fognini, the world number 45, was beaten in the two sets on the clay at Parque Sarmiento in Buenos Aires.
Flavia Pennetta defeated Roberta Vinci earlier this year in the first ever all-Italian Grand Slam final to claim the biggest win of her career - the US Open, and then announced her retirement from the sport
Rafael Nadal had been beaten twice previously this season by the experienced Italian eighth seed Fabio Fognini, in Rio de Janeiro and then in Barcelona.
Fabio Fognini is a familiar foe for top seed Rafael Nadal, having already beaten the Spaniard twice this season in Rio de Janeiro and Barcelona.
Unseeded Italians Simone Bolelli and Fabio Fognini, who were in their ninth Grand Slam as a team, battled to a 6-4, 6-4 win in 1hr 22mins against
Italy's No. 17-ranked Fabio Fognini showed his middle finger to the crowd as he left the stadium following his 7-6 (5), 6-4 loss to 553rd-ranked Chinese wild card Wang Chuhan in the first round on Tuesday. He also gave Wang a shove with his elbow after the two shook hands at the net.
After being beaten by 553rd-ranked Wang Chuhan in the first round, Fabio Fognini gave Wang a little shove with his arm as they crossed paths, then flashed his middle finger to the crowd upon exiting.
Wang Chuhan, who mainly plays in Challenger or Futures tournaments rather than the top tier, recorded a 7-6(5) 6-4 first round win over the Italian on the Grandstand court to huge cheers from the shocked home crowd.