This time, he had company, as safety Eric Reid, who was not in uniform, joined him in taking a knee just before the anthem began as boos rained down from the crowd.
On Sunday, Kaepernick vowed to continue his protest during the national anthem until "significant change" is made regarding racial oppression in the United States.
After sitting during the anthem in the first three preseason games, Kaepernick followed through on that promise again Thursday night at Qualcomm Stadium.
"Yes, I'll continue to sit," Kaepernick said earlier this week. "I'm going to continue to stand with the people that are being oppressed. To me this is something that has to change. When there's significant change and I feel like that flag represents what it's supposed to represent, this country is representing people the way that it's supposed to, I'll stand."
Kaepernick sat on a cooler away from the team in the previous games. On Thursday, he was among his teammates on the sideline. By his side was Reid, a native of Baton Rouge, Louisiana. Reid has been outspoken about police brutality in the wake of the shooting death of Alton Sterling in that city and the impact it has had on him.
One person who was standing during the anthem on the 49ers sideline was Nate Boyer, the former Green Beret and Seattle long-snapper, who earlier in the week penned a letter to Kaepernick on armytimes.com.
Boyer told ESPN that he and Kaepernick had never met before Thursday, but Kaepernick invited him to come to the game after reading Boyer's letter. Boyer and Kaepernick had a conversation before the game and exchanged their views and ideas on Kaepernick's stance.
Boyer is working with Fox Sports' Jay Glazer, who first reported Boyer's attendance, on a charitable foundation called Merging Vets & Players (MVP). He traveled from Los Angeles to meet with Kaepernick and attend the game.
Before the game, the Chargers kicked off their 28th annual "Salute to the Military" with 240 military members presenting a "super flag" and color guards from each of the four major military branches presenting the regular-sized flag.
Petty Officer 1st Class Steven Powell sang the anthem. He declined to discuss Kaepernick's protest but said he was "grateful" for the opportunity to perform in front of so many of his military brethren.
At the stadium, the Chargers played Lee Greenwood's "God Bless the USA" in the first half and asked military members in attendance to stand. When they did, Kaepernick stood and applauded with his teammates and the crowd for the veterans.
While many have drawn the connection between Kaepernick's protest and the military, the quarterback has insisted that his protest has nothing to do with that.
"I have great respect for the men and women that have fought for this country," Kaepernick said on Sunday. "I have family, I have friends that have gone and fought for this country. And they fight for freedom, they fight for the people, they fight for liberty and justice, for everyone. That's not happening.
"People are dying in vain because this country isn't holding their end of the bargain up, as far as giving freedom and justice, liberty to everybody. That's something that's not happening. I've seen videos, I've seen circumstances where men and women that have been in the military have come back and been treated unjustly by the country they fought for, and have been murdered by the country they fought for, on our land. That's not right."
Earlier in the day, Kaepernick took to social media to address the burgeoning story about his wearing socks that depicted pigs wearing police hats during training camp practices.
"I wore these socks, in the past, because the rogue cops that are allowed to hold positions in police departments, not only put the community in danger, but also the cops that have the right intentions in danger by creating an environment of tension and mistrust," Kaepernick wrote on Instagram. "I have two uncles and friends who are police officers and work to protect and serve ALL people. So before these socks, which were worn before I took my public stance, are used to distract from the real issues, I wanted to address this immediately."
Kaepernick, who missed the first two preseason games because of right shoulder tightness, made his first start of the preseason against the Chargers. Niners coach Chip Kelly said on Tuesday that he was hoping to get a longer look at Kaepernick in San Diego because he'd played only 13 snaps in the preseason.
Information from ESPN's Paul Gutierrez was used in this report.