Antonio Conte has explained that three of his players who weren't included in the matchday squad against Brighton will also miss out against Southampton.
Spurs played Graham Potter's side in the FA Cup on Saturday and won 3-1, taking them to the next round of the competition, where they will travel to Middlesbrough.
The busy fixture schedule doesn't stop for the Lilywhites and they will have had just a few days to prepare for their next game, which is a return to the Premier League after the winter break.
Before the break, Tottenham lost their third game of the month to Chelsea but since then have welcomed Rodrigo Bentancur and Dejan Kulusevski to their side, as well as seeing four first team departures.
The Brighton game saw Cristian Romero and Son Heung-min return to the starting line-up after their injuries, with the formers being a longer-term one which has meant he hasn't got much game time under his new boss.
There was hope that Tottenham would return to their fixture schedule with a fully fit squad and a boost from their new arrivals.
However, Eric Dier who was carrying an injury before the break, Oliver Skipp and Japhet Tanganga were all left out of the matchday squad completely.
Dier has become a key part of the Italian's side in recent times and although he returned to the starting XI at Stamford Bridge, he had heavy strapping on his leg throughout the game.
The trio were all left out of the squad on Saturday and Conte has revealed that it will be the same case in Wednesday's match at the Tottenham Hotspur Stadium.
"No news. Eric Dier is not available and it's the same for Skipp and Tanganga. The others are all available," he admitted in his pre match press conference.
No new injuries means the Italian will have most of his squad available against Ralph Hasenhuttl's side, but he will again not have his completed back three due to the 28-year-old's absence.
"He [Dier] had the same problems in the past before to be able to play the last game against Chelsea," Conte explained.
"He had a little muscular problem and it's not serious but it's in the same place as that problem. The doctor said not to use him and that's a pity because you know he's an important player."
Skipp's absence may have come as a bit of a surprise to some and the Italian provided some details of his injury, ahead of the Southampton game.
"He had a problem then we tried to take care with him then we need to have a bit of patience with him," Conte said. "He is another player that I have seen great improvement in him. It's a pity that he is unavailable.
"I hope to have him back very soon. The medical department told me two weeks but I hope to have him available in one week/ten days maximum and for this reason I push the medical department because we're talking about an important player for Tottenham Hotspur."