That was the second time it has happened recently, but O'Neill insisted Heskey's fitness is not a major cause for concern.
"Emile came off again against Blackburn with a tight hamstring to prevent any further damage," said O'Neill. "But he is now not feeling too bad.
"I think that sometimes in the case of Emile, when you haven't played for a little while, there is a possibility that the hamstrings are tight. It is something that a few players have complained about recently.
"But Emile in particular was suffering from it and there was no point in risking him. From that viewpoint we made the substitution.
"He did not complete the previous game in the FA Cup but I am hoping that it is not too big a problem."
Heskey is poised to start against West Ham as Villa look to exact revenge for their last-gasp defeat at Upton Park in November.
And O'Neill does not think the Hammers' off-field issues will have any effect on their players.
He added: "The game against West Ham is naturally difficult for us.
"They will come to Villa Park and play strongly. The off-the-field goings on at the moment will not affect their players."
O'Neill is an admirer of West Ham boss Gianfranco Zola.
He said: "Zola has been a really great player and he is coping with things magnificently in management.
"These things are set to try him out and he is going along very well. This is why West Ham will be strong on the day."
O'Neill is looking for Villa to build upon their 1-0 win at Blackburn as he prepares his team for three games in six days.
He is particularly keen for his side to get back to winning ways in the league following defeats to Liverpool and Arsenal.
"We could have had a more conclusive win at Blackburn," he said.
"There were a couple of decisions which went against us. They are very important and in the scheme of things they might prove even more important.
"We had had a little break before the game and I thought we looked very fresh at Blackburn.
"Some of our first half play was very inventive indeed considering it was a semi-final with a few nerves jangling around the place.
"But Sunday's game against West Ham is just as important, every bit as important as we want to regain momentum in the league.
"Obviously that was lost with the defeats against Liverpool and Arsenal. The Liverpool defeat was particularly galling.
"We have to fight back and put aside any thoughts of Wednesday night because the Premiership is very, very important."