The central defender picked up a foot injury in the Champions League two and a half weeks ago and has been left out by Spain for the World Cup qualifiers against Belarus on Friday and France four days later.
Pique returned to training with the Barca squad on Saturday afternoon but Vilanova was cautious about rushing him back for a high-profile match against Real.
"Pique has been out of action for quite a few days," Vilanova told a news conference.
"If he had at least trained in this time we would have more information and I am not in favour of (painkilling) injections because this could give us problems further down the line.
"There is no doubt about Pique's willingness to play because he has dedicated all the hours possible to recover but we cannot fight against nature," added Vilanova.
Barca are already without influential centre half Carles Puyol who dislocated his elbow during the week.
Vilanova is most likely to play midfielders Javier Mascherano and new signing Alex Song in the middle of the defence on Sunday.
The duo have looked uncomfortable when thrown together over the last couple of weeks.
Barca have won all six league games and already hold an eight-point lead over Real who only dropped 14 points on their way to the title last season.
Sunday's game has been given added significance because of the growing political debate within Spain over Catalonia's desire for independence.
Barca, often seen as a symbol of the independence movement, are planning to turn the Nou Camp into a giant red and yellow Catalan flag but Vilanova, like his opposite number Jose Mourinho earlier in the day, refused to get drawn into the debate.
"There are always Catalan flags and mosaics here, they have done it many times before," Vilanova said.
"When we go to other stadiums we see other flags and it isn't a problem. It is only a game of football."