Nearly 200 players raised their right fists and took a knee to express solidarity with the Black Lives Matter movement before the MLS is Back Tournament opener on Wednesday in Orlando, Florida.
Some players wore t-shirts over their jerseys with the words "Black and Proud" and "Silence is Violence" before the match between Orlando City and Inter Miami, while both starting line-ups, as well as the referees, took a knee just before kick-off.
The on-field demonstration lasted eight minutes and 46 seconds, the amount of time a Minneapolis police officer knelt on the neck of George Floyd, the unarmed American black man whose death in May sparked worldwide protests.
Toronto FC's Justin Morrow, executive director of the Black Players for Change organisation, told ESPN at half-time: "This is a chance for us to take our place with them, stand up against racial inequality, fight that battle and make sure our voices in Major League Soccer are heard.
"What you saw tonight was a special moment that was driven by the players, and Major League Soccer collaborated with us to make that happen."
Black Players for Change is an organisation of more than 170 black MLS players that was formed to give them a voice and assist in making systemic change both inside and outside of the league.
Former Manchester United winger Nani, who scored the winner for Orlando deep into stoppage time, said: "It's a beautiful gesture from everyone.
"We all know we want to change the world, we want a better world, in which people look to each other without any difference, with no discrimination for colour or nationality and I think the decision at the beginning of the game and the performance from everyone was beautiful.
"It's emotional for those there and I felt it for a couple of moments and I started thinking that everyone in the world should stop for a minute and start thinking about their attitudes and to see what we all can do to make a difference in the world.
"We must think about our children and the future for them, to teach them to behave, to be a better person and to create a better world."
The kneeling protest was popularised by then-San Francisco 49ers quarterback Colin Kaepernick in 2016 while the raised fists mirrors the protest against racial inequality by Americans John Carlos and Tommie Smith at the 1968 Mexico Olympics.
Premier League players have knelt at the beginning of every match since English football's top flight resumed following the coronavirus lockdown.
The MLS is Back Tournament is a one-off competition during the 2020 Major League Soccer season to mark the league's return to action from the coronavirus pandemic, with all matches being held at the ESPN Wide World of Sports Complex.
Expansion team Miami, part-owned by former England captain David Beckham, went ahead early in the second half through Juan Agudelo but the game changed when their defender Andres Reyes took an elbow to the face and was taken off on a stretcher.
Nani passed to Christopher Mueller for the equaliser in the 70th minute before the Portuguese delivered the game winner seven minutes into stoppage time.
But the tournament has had a stuttering start. All 26 MLS clubs were set to take part until FC Dallas were withdrawn on Monday after 10 players and a staff member tested positive for the coronavirus.
Nashville could follow Dallas after five players tested positive and four more required further testing. On Tuesday, the Vancouver Whitecaps announced five members of the team would not make the trip to Orlando due to coronavirus concerns.
Those same concerns led LAFC's Carlos Vela, the league's reigning MVP, to opt out of the tournament on Monday.