The US President said his comments about national anthem protests have “nothing to do with race” as he demanded players from Jackson Jaguars and Baltimore Ravens show “respect” for the US and its flag.
Players took to their knees on Sunday in an act of defiance against Donald Trump after the American leader urged people to boycott the league, accusing athletes of disrespecting the national flag.
And the players who did not kneel at Wembley linked arms as the American national anthem was played.
Some angry fans booed their own teams and even burned their shirts in retaliation.
But Mr Trump hit back at the dramatic scenes at Wembley Stadium.
He tweeted: “Many people booed the players who kneeled yesterday (which was a small percentage of total). These are fans who demand respect for our Flag!
“The issue of kneeling has nothing to do with race. It is about respect for our Country, Flag and National Anthem. NFL must respect this!”
Mr Trump later tweeted the hashtag #StandForOurAnthem, which has now gone viral on social media.
The US President stepped up his feud about players who refuse to stand for the anthem in what began last year as a protest over excessive use of force by police against black Americans.
It comes after he was heavily criticised for appearing to say anti-racism demonstrators were just as bad as far-right activists following a white supremacists rally in Virginia in August.
NFL player Colin Kaepernick first demonstrated during the anthem in preseason back in 2016.
He said: ”I am not going to stand up to show pride in a flag for a country that oppresses black people and people of colour.”
“To me, this is bigger than football and it would be selfish on my part to look the other way.”
But the row escalated on Friday when Mr Trump suggested any protesting football player was a “son of a b***h” and should lose his job.
At a political rally in Alabama, he asked a crowd of supporters: “Wouldn’t you love to see one of these NFL owners, when somebody disrespects our flag, to say, ‘Get that son of a bitch off the field right now… he is fired’?”
But dozens of players flouted the tradition this weekend following his comments in a direct act of defiance.