Here is every moment Holt weighed in on:
ON TAXES AND EMAILS
"Mr Trump, we're talking about the burden that Americans have to pay, yet you have not released your tax returns," said Holt.
"Don't Americans have a right to know if there are any conflicts of interest?"
When Trump said he would release his tax returns after an audit, Holt pointed out that he could do so right now.
Trump: "Almost every lawyer says, you don't release your returns until the audit's complete. When the audit's complete, I'll do it. But I would go against them if she releases her emails."
Holt: "So it's negotiable?"
Trump: "It's not negotiable, no. Let her release the emails. Why did she delete 33,000..."
Holt: "Well, I'll let her answer that."
When Clinton didn't answer the question in her reply, Holt pushed her this far: "He [Trump] also raised the issue of your emails. Do you want to respond to that?"
ON RACIAL DIVIDES
When Trump talked about the importance of "law and order" in healing America's racial divide, Holt interjected that stop-and-frisk had been ruled unconstitutional and that it had been called "a form of racial profiling".
He did then ask Clinton if she believed the police were "implicitly biased against black people", but quickly turned back to Trump to quiz him on the Barack Obama "birther" conspiracy.
"For five years, you perpetuated a false claim that the nation's first black president was not a natural-born citizen," said Holt.
"You questioned his legitimacy. In the last couple of weeks, you acknowledged what most Americans have accepted for years: The President was born in the United States. Can you tell us what took you so long?"
And he pressed the (admittedly important) point: "I just want to get the answer here. The birth certificate was produced in 2011. You've continued to tell the story and question the President's legitimacy in 2012, '13, '14, '15..."
And again: "We're talking about racial healing in this segment. What do you say to Americans, people of colour..."
ON IRAQ WAR
Holt then entered an argument with Trump over whether he supported the Iraq War - the businessman claims he was an early opponent, although his comments at the time were rather ambiguous and he gave lukewarm support for the invasion in 2002.
Meanwhile, Clinton supported the war.
"Why is your judgment any different than Mrs Clinton's judgment?" asked Holt.
ON CLINTON'S APPEARANCE
The moderator quizzed Trump on an insult towards Clinton.
"Earlier this month, you said she doesn't have, quote, 'a presidential look'. She's standing here right now. What did you mean by that?" he asked.
THE REACTION
Holt received mixed reviews from social media and the global press, much of it based on that person or publication's political leanings.
Twitter dubbed Holt the invisible man, asking where he had gone and why he wasn't moderating.
Right-wing daily The Washington Times gave Holt an "F" grade, saying he was "anything but moderate", giving Trump "the most pointed questions all night" and not questioning the Democratic nominee on the Clinton Foundation pay-for-play allegations, or FBI investigation into her emails.
TVLine noted that Holt's questions on race and birtherism might lead Trump supporters to accuse him of pro-Clinton bias. "But voting records prove Holt is a registered Republican, so it's not as if he's a diehard liberal. Bottom line: Moderating this debate was a thankless job, but Holt performed admirably under considerable fire."
Slate praised Holt for asking "good, tough, smart questions" while Fortune said Holt "saved his fact-checking for a few, spare moments in the debate, and he mostly focused on Trump's statements."
Fox News host Megyn Kelly said Holt did a "fine job" and conservative radio host and Trump supporter Hugh Hewitt praised the moderator for giving Trump "plenty of time" to answer questions.
But former New York Mayor Rudy Giuliani criticised Holt for getting involved in a stop-and-frisk debate he knew nothing about and not interfering on Trump's behalf.
The Republican nominee himself had a perhaps surprising view.
"I thought Lester Holt did a great job," said Trump. "I thought the questions were very fair."
