Mr. Biden, however, forgetting about unity for a spell, was not content to let the fight go.

“I’m prepared to compare my foreign policy credentials up against my friend here on any day of the week and every day of the week,” he said.

Perhaps it was unrealistic for Biden allies to expect much deference from Mr. Sanders — and for anyone to expect Mr. Biden, rarely cited for his discipline, to play the reserved statesman all night.

During a news conference on Wednesday, a day after Mr. Sanders suffered a second night of big primary losses, he registered an ideological wish list of policy issues as if to offer Mr. Biden a record of concessions he might seek if he were to drop out.

“What are you going to do?” Mr. Sanders asked Mr. Biden again and again from a lectern at a hotel in Vermont, challenging his rival to address the systemic problems he has long placed at the heart of his progressive agenda.

In the earlier days of the contest, such a head-to-head matchup like the one Sunday night would have been a dream scenario for the Sanders campaign. Aides have long viewed Mr. Biden, an establishment moderate, as the perfect foil for Mr. Sanders’s promise of a political revolution. They have often ached for Mr. Sanders to challenge Mr. Biden more directly.

It may now be too late. Though there were previous flashes of confrontation, it was not until recent days that Mr. Sanders began to aim squarely at Mr. Biden’s vulnerabilities.

But if Mr. Sanders will always fight for his agenda, as his long history of doing so has made clear, the debate may be the last time Mr. Sanders faces Mr. Biden as an opponent. Though he has not given any indications he will leave the race — and he and his team say he is still running to win — the upcoming primary calendar, which includes states like Florida and Ohio on Tuesday, does not exactly play to his strengths. The goal may now be as much about accumulating delegates to use as leverage in negotiations with Mr. Biden as it is about winning.