The day after Ryan, the top Republican in Congress, told party lawmakers he was breaking with the presidential nominee and would not campaign for him, Trump issued a barrage of social media posts criticizing Republicans who have fled his campaign.
The stinging attacks deepened a dramatic rift in the party over the former reality TV star, who has seen a string of Republican defections after a video surfaced on Friday showing him bragging crudely to a reporter in 2005 about groping women and making unwanted sexual advances.
"Our very weak and ineffective leader, Paul Ryan, had a bad conference call where his members went wild at his disloyalty," Trump said in a tweet on Tuesday.
He complained in another tweet that it was hard to do well with "zero support" from Ryan and others, but added in a later Twitter post, "It is so nice that the shackles have been taken off me and I can now fight for America the way I want to."
Ryan told congressional Republicans he would put his energy into preserving party majorities in Congress, all but conceding that Democratic candidate Hillary Clinton would likely win the White House in the Nov. 8 election. The move angered some Trump supporters, although Ryan said he would not withdraw his endorsement of the New York businessman.
Trump, 70, has portrayed himself as tough on national security, and his campaign released a television advertisement on Tuesday featuring footage of Clinton, a 68-year-old former secretary of state, stumbling last month after leaving a service commemorating the Sept. 11 attacks. Her campaign said she had been diagnosed a few days earlier with pneumonia.
"Hillary Clinton doesn't have the fortitude, strength or stamina to lead in our world," the ad's narrator says. "She failed as secretary of state. Don't let her fail us again."
Trump, whose campaign has been marked for months by controversies over both his policies and his brash style, has slipped further behind Clinton in opinion polls.
Many Republicans are worried his chaotic campaign could hurt their chances of holding majorities in the House of Representatives and Senate in next month's election, and will inflict long-term damage on the party.
In an extraordinary party revolt, nearly half of all 331 incumbent Republican senators, House members and governors have condemned Trump's lewd remarks on the video, and roughly one in 10 have called for him to drop out of the race, a Reuters review of official statements and local news coverage indicates.
Unlike Ryan, Republican National Committee Chairman Reince Priebus told RNC members on Monday that the committee, the party's leadership and fundraising arm, still backed Trump, two RNC members told Reuters.
Trump's vice presidential running mate, Indiana Governor Mike Pence, reinforced his support in television interviews on Monday.
New Jersey Governor Chris Christie, a former rival for the White House who has became a close ally of Trump, also reaffirmed his backing although he called Trump's comments in the 2005 video "completely indefensible."
"In the end, this election is about bigger issues than that," Christie told CBS Radio's WFAN on Tuesday, adding he did not blame other Republicans who have withdrawn their support.
U.S. Representative Steve King of Iowa, however, did not hesitate to criticize those backing away from Trump.
"That's a mistake ... that drags the entire ticket down," he said in an interview with CNN. "What does it do to your integrity if you help Hillary Clinton become president?"
Both Trump and Clinton head to the key battleground state of Florida with campaign events later on Tuesday.
The Reuters/Ipsos State of the Nation project released on Monday estimated that Clinton had at least a 95 percent chance of winning the 270 Electoral College votes needed to become president. The polling did not capture reaction to Trump's performance in Sunday's debate or Friday's news reports on the video.
Clinton, the first woman to be picked as a presidential nominee by a major U.S. party, tried to focus on issues that could win over undecided women voters on Tuesday, proposing to expand the child tax credit so more low-income families can benefit and to increase by $1,000 a year the tax credit available to families for each child up to age 4.