Colin Kaepernick, San Francisco 49ers' back-up quarterback, brought his protest to draw attention to racial inequality to a nationally televised Monday Night Football audience, by kneeling on the field rather than standing as the national anthem played.
He was joined on the sidelines by 49ers safety Eric Reid, who knelt beside Kaepernick, while two other teammates, safety Antoine Bethea and linebacker Eli Harold, stood with fists raised during the performance of the Star Spangled Banner.
While military personnel unfurled a giant American flag over the field, a number of fans shouted at Kaepernick "Why don't you stand up?", according to an ESPN commentator reporting for the Disney-owned sports channel's broadcast.
At least two players from the opposing team, wide receiver Kenny Britt and defensive end Robert Quinn of the Los Angeles Rams, also held their fists in the air for the opening ceremony.
Several 49ers squad members also rallied around the smiling Kaepernick after the anthem to show their support.
The protests have not been popular with law-enforcement officers who were broadly categorised by Kaepernick as "getting away with murder".
The local police union even suggested officers might boycott their voluntary security duties at the Monday night game, but that has not materialised.
"To make that kind of blanket statement, for all police officers, it's disheartening," Lieutenant Dan Moreno, of Santa Clara Police Department, told Al Jazeera.
"We may not like it ... but we realise that's his right to have an opinion."
"This was the first weekend of the American Football season. In stadiums across the country there were echoes of Colin Kaepernick's national anthem protest," said Al Jazeera's Allen Schauffler, reporting from California.
"What started as an individual statement has spread and grown and, while hardly universal, shows no sign of dying out."
Kaepernick has ignited controversy within the National Football League through an act of defiance he introduced during pre-season games to protest against what he has called racial injustice and police brutality.
"One injustice against one American ... is an injustice against all Americans. We should all get behind that," said Daryl Graves, 52, a fan who was in the stands during the game in San Francisco.
"Change doesn't happen without confrontation."
The gesture, which went largely unnoticed initially, has since been imitated by numerous other NFL players.
On Sunday, Kansas City Chiefs cornerback Marcus Peters, also a black, raised his fist during the national anthem for his team's home-game season opener against the San Diego Chargers.
His action recalled the raised-fist salute by black athletes Tommie Smith and John Carlos during their medal ceremony at the 1968 Olympic Games in Mexico City.
Carlos, now 71, praised Kaepernick, Peters and other NFL players in a Reuters interview on Monday for using their televised platforms as professional athletes to give Americans a "shock treatment".
Other members of the Chiefs linked arms during Sunday's anthem, as did the Seattle Seahawks at their home game against the Miami Dolphins. The exact meaning of their gestures was not clear.
Four members of the Dolphins knelt while The Star-Spangled Banner played on Sunday. In Thursday's NFL season opener, Denver Broncos linebacker Brandon Marshall had knelt during the anthem.
The protesting players have been seen as allies of the Black Lives Matter movement, which grew in response to a series of police killings of unarmed black people across the country.
About two-thirds of NFL players are black.
The protests also have provoked anger in some fans who see the gesture as disrespectful to the US flag, the military and the nation in general.
President Barack Obama has weighed in on the controversy, saying Kaepernick was exercising a constitutional right and provoking conversation "around some topics that need to be talked about".
"The start of the American football season this year corresponds with the 15th anniversary of the 9/11 terrorist attacks, said Al Jazeera's Schauffler. "So national unity and respect for the flag are particularly sensitive issues.
"Kaepernick, who will make more than $11m this year, says he's speaking for those who don't have his kind of platform."