James McCarthy is one of four Republic of Ireland players who has been forced to withdraw from the final squad for Saturday's trip to Austria.
The Everton midfielder has a hamstring injury and joins Cyrus Christie, Stephen Ward and Stephen Quinn in missing out owing to fitness issues.
Toffees boss Ronald Koeman last week urged McCarthy to skip international duty.
"The player is not fit and not involved in team sessions," said the Dutchman. "To have a call-up he needs to be close to being fit, but it's a bit of the same story as to what happened last time.
"We spoke to James and my advice to him is to stay here, train and get fit as soon as possible. But the final decision is down to the player."
O'Neill named a provisional squad last Wednesday, when Shane Long's exclusion - also for a hamstring problem - was confirmed.
The list does include uncapped duo Daryl Horgan and Andy Boyle, who have impressed in Dundalk's Europa League campaign after helping the club to regain their domestic title.
Ireland are level with Serbia on seven points at the top of Group D, having played three matches.