Players from he Jacksonville Jaguars and Baltimore Ravens refused to stand as the national anthem rang out at the London venue.
Their actions came after President Trump urged people to boycott the league, accusing athletes of disrespecting the national flag.
People who did not kneel linked arms as the anthem was heard.
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The players kneeled during the anthem
In an early morning post on Twitter, Mr Trump 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.
He tweeted: ”If NFL fans refuse to go to games until players stop disrespecting our Flag & Country, you will see change take place fast. Fire or suspend!”
In a second tweet, Mr Trump said that the “league should back” fans who are upset about the protests.
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It was part of an ongoing protest
As the London game began, the Ravens tweeted a message saying: “We recognise our players’ influence. We respect their demonstration and support them 100 per cent.
“All voices need to be heard. That’s democracy in its highest form.”
The Jaguars shared a picture of team owner Shad Khan arm-in-arm with his players and the caption “Unity”.
In 2016, then San Francisco 49ers quarterback Colin Kaepernick put one knee to the ground during pre-game renditions of the “Star Spangled Banner”. Several players have since made similar gestures of protest before games.
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Mr Trump is in a row with top sports stars
At a political rally in Alabama on Friday, Mr Trump suggested any protesting football player was a “son of a bitch” and should lose his job.
On Sunday morning, New England Patriots Chairman and CEO Robert Kraft criticised Mr Trump and defended players’ right to protest.
He said: ”I am deeply disappointed by the tone of the comments made by the president on Friday. Mr Kraft said he supported his players’ “right to peacefully affect social change and raise awareness in a manner they feel is most impactful.”
NFL Commissioner Roger Goodell said Trump’s comments in Alabama revealed an “unfortunate lack of respect” for the NFL and its players.
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Donald Trump says athletes are disrespect the flag
National Basketball Association players also struck back against comments by the president on Saturday after Trump clashed with one of the biggest stars in the NBA.
In an early morning Twitter message on Saturday, the president rescinded a White House invitation to Stephen Curry, who had said he would “vote” against the planned visit by the NBA champion Golden State Warriors.
Mr Trump tweeted: ”Going to the White House is considered a great honor for a championship team. Stephen Curry is hesitating, therefore invitation is withdrawn!” Trump tweeted.
Curry told a news conference in Oakland, California: “It’s beneath the leader of a country to go that route.”
The Oakland-based Golden State Warriors said in a statement the team had intended to meet to discuss the potential visit at the first opportunity on Saturday morning.
Singer Stevie Wonder appeared to evoke protests by Kaepernick and other athletes when he put one knee to the stage during a concert at the Global Citizen Festival in New York City on Saturday.
Wonder told the audience: “Tonight, I’m taking a knee for America,”
He added: ”I’m taking both knees.
”Both knees in prayer for our planet, our future, our leaders of the world and our globe.”