Their League Two opponents are playing a Premier League side for the second time in quick succession after competing at Watford at the weekend.
With many of the club's key players still taking time off after representing their nations at Euro 2020, Nuno Espirito Santo remains relatively unsettled at Spurs at a time that he is looking to make an instant impact.
Nevertheless, given the possibility of Harry Kane's departure this summer, it has provided the Portuguese with a chance to take a glimpse at life without the club's star player.
Top prospect Dane Scarlett emerged as the biggest plus from Saturday's friendly at Leyton Orient with the 17-year-old getting on the scoresheet in the first half.
The teenager's first-team opportunities are likely to remain limited during the upcoming campaign, but it at least gave supporters further hope that they will eventually have a homegrown replacement for Kane in the future.
Given the youngsters present during both the first and second halves, Nuno would have been relatively pleased with the team's first run-out of the summer.
However, with their Premier League opener against Manchester City now less than four weeks away, he will want his squad to up their level against another team from League Two.
Colchester endured a desperately disappointing 2020-21 campaign, finishing in 20th position in the fourth-tier standings and losing to non-league Marine in the FA Cup.
Promoting Hayden Mullins to the role of head coach has had a positive effort, however, with the former midfielder ending the season with 11 points from eight matches.
That progress was carried over into Saturday's trip to Watford as his team lost just 2-1 and 2-0 respectively to the Hornets in two 60-minute contests.
The result on Wednesday will not matter in the long run, but Mullins will feel that there is a realistic chance of an upset given the level of player he has managed to attract to the Colchester Community Stadium.
However, more first-team players may be in a position to feature after no risks were taken on them at the weekend.
Head To Head
Each of the three competitive encounters between these teams have been played since 2016, starting with Spurs posting a 4-1 win in the FA Cup.
However, Colchester got their revenge in 2019 with a penalty-shootout triumph in the EFL Cup after holding their more illustrious opponents to a goalless draw.
Two weeks later, a fixture in the EFL Trophy ended in a 1-1 draw.
We say: Colchester United 0-2 Tottenham Hotspur
Having given a good account of themselves against Watford, Colchester will feel that they can test a depleted Spurs outfit.
Nevertheless, the North Londoners should still have enough quality in their ranks to run out comfortable winners.