The news represents a fresh concern for manager Zinedine Zidane with Gareth Bale, Dani Carvajal, Mateo Kovacic and Keylor Navas also remaining on the sidelines due to different injuries.
Isco, 25, was seen limping down the tunnel after completing the full 90 minutes of Real's 2-1 defeat at Girona on Sunday (29 October).
The Spaniard confirmed after the disappointing game at the Estadi Montilivi that he did suffer a knock in the second-half but he was confident on recovering in time to face Tottenham.
"I have a problem. I've been hit, but by Wednesday I'll be back," Isco said to the reporters.
Isco's absence from the workout only two days before the Champions League clash might be just precautionary but Varane's unavailability to complete the training with the rest of his teammates looks like being a bigger concern for Zidane.
The France international had to be replaced by Nacho at half-time during the defeat to Girona and missed matches earlier in the season due to another muscle injury.
The potential absence of Isco and Varane for the clash with Tottenham would be a major blow for Zidane as both players are guranteed starters.
Marco Asensio could replace Isco at Wembley if the former Malaga star doesn't recover on time while Nacho should partner Ramos in the heart of the back-line in case Varane misses the game.
Meanwhile, the trip to Tottenham is expected to come too early for Bale, Dani Carvajal, Mateo Kovacic and Keylor Navas with Real confirming that the four players are still working on their recovery from different fitness issues.
Bale, Carvajal and Kovacic missed the draw with Spurs at the Santiago Bernabeu on 17 October while Navas is yet to play a game since then due to a thigh injury.
"The squad trained at Real Madrid City as they begin to prepare for the Champions League game against Tottenham, which will be played on Wednesday at Wembley (8.45pm CEST). The players who started the game against Girona trained at a lower intensity out on the field, apart from Isco and Varane, who worked inside," Real Madrid confirmed in the club website.
"Zidane had the Castilla goalkeeper Moha and the under-19s Darío with him in a session where ball work was the focus. The madridistas carried out ball circulation exercises and pressing, shooting and matches in reduced sized pitches. Bale, Carvajal, Kovacic and Keylor NavasThe first two trained alone on the field with and without a ball."