While first place in Group D was still up for grabs, several Spain players looked fatigued in the latter stages of the match in Bordeaux, with Ivan Perisic striking an 87th minute winner.
But Del Bosque insists he had confidence in the condition of his players, and believes shaking things up will not have had a huge difference.
"I think they may be tired. That's normal playing a game in this temperature against this team, but I think we were in good condition to play this game," he said.
"We don't have a scientific way of saying these [players] can play, or these can't play.
"We have lots of confidence in the physical condition of our players."
The defeat to Croatia means Spain face Italy in the round of 16, but Del Bosque is not concerned about facing the one-time winners.
"I think we have time to analyse what that means against Italy. I think it wasn't the path we wanted to take," he said.
"We looked like we had controlled [the game against Croatia] but football's brought us to the situation and we will try to recuperate as good as possible and be in the best possible condition on Monday.
"They're playing well, before today we were playing well, and even today we haven't played badly. We have to prepare to play against Italy."
Del Bosque also came out in defence of goalkeeper David De Gea, who was beaten at his near post for the winning goal.
"He didn't have too much to do and we can't blame him for the goals. The second was a counter attack the first was from close range," the 65-year-old added.
"I don't think we have to blame anyone specifically. We've lost, all of us have lost, we're all to blame."