Like the previous season's Ex-Factor, this season also had a twist theme. It was called Project Do Not Assume or Project DNA for short and had two aspects. The first was that two of the contestants, Michael and Jennifer were half-siblings who were unaware of each other before the show. They discovered this in the second week and revealed it to the other houseguests.
The second Project DNA aspect was a pair of identical twins who switched places every few days, appearing as one person, Adria, to their housemates. If they were not discovered after five weeks in the house, they would be able to live in the house simultaneously as individuals. The twins evaded eviction in the fourth week when Adria was nominated as a veto replacement by the Head of Household, Drew. Host Julie Chen introduced Natalie to the other houseguests on Day 35, minutes following Scott's eviction.
The Golden Veto was tweaked slightly to increase the possibility that the veto would be exercised. From this season on, only a maximum of six players could participate in any veto contest: the two nominees and the Head of Household all getting to select one person each. The winner was free to use or decline to use the veto as he or she saw fit, but with so few players playing, there were indeed more vetoes this season than in the previous two seasons combined.
A new strategic move known as backdooring also originated during this season. Backdooring occurs when a Head of Household puts two players up for nominations whom the Head of Household actually doesn't have any intention to get evicted. Instead, the Head of Household and the two nominees have agreed to band together to rid the Big Brother house of a common threat. During the veto challenge, these three players only choose players whom they feel will use the veto if it is won (the common threat isn't given a chance to participate in the Power of Veto challenge). The player who won the veto uses it, and the common threat goes up for nomination and gets evicted.
Backdooring has greatly impacted on how this game was played. During this season, the 'Four Horsemen' alliance (Jase Wirey, Michael Ellis, Drew Daniel, and Scott Long) dominated the Big Brother house for weeks until Diane Henry won Head of Household. Diane attempted to evict Jase during her reign, but she failed when he won the veto and removed himself from the nomination block. The next week, Jennifer (also known as "Nakomis") won Head of Household. Jennifer put a plan into motion that would later become known as backdooring. This specific plan was called the Six-Finger Plan when it was first conceived. Jennifer put Diane and Marvin up for eviction without any intention of getting them evicted. Instead, Jennifer's plan was to finally get Jase evicted from the house. Drew won the veto, and he used it to take Diane off the nomination block. Jase figured out what had happened before being nominated; however, by then, it was too late. Michael Ellis had said to Jase as they figured out the scheme, "You just got bitch slapped." Jase became nominated and was evicted by a vote of 6-1. Later on in the season, another backdooring plot that Diane created led to Karen's eviction.
An additional twist was a "Double Elimination Week" in the tenth week of the game, in which the entire process of selecting a Head of Household and veto holder, and evicting a player all occurred twice in one week.
Adria and Natalie were already considered a big threat before Adria made her bold move. Even her own alliance started questioning her, saying that the twins were "solid" and they know they can count on each other. When Adria won HoH, after revealing that Natalie was her twin, she put up Marvin (a "floater") in the game, and Will (from her alliance). Adria's plan was to form 3 smaller alliances in the game, breaking up the two existing alliances. Everyone was stunned by this, Adria's plan backfiring by uniting the rest of the house to try and evict the twins. Adria won the Power of Veto, and despite Will's hopes that she would use the Veto to save her alliance; she did not, meaning that Will was evicted and was the first person to be sent to the jury house. Afterwards, the power shifted and Adria and Natalie were nominated for eviction. In the veto competition, the twins showed their power when Adria won the power of Veto for the second week in a row. When the Veto was used, Adria chose to save herself. Natalie was evicted, and Adria was without allies. After losing the HoH competition, Adria was put up again. In the veto competition, Adria almost won but lost to Karen, Karen's first victory in the game so far. Adria tried to convince Karen to save Adria, claiming that Karen was a pawn in her friend's group and was considered useless. Karen told Adria that she wanted to save her, but didn't want to break the promise of her alliance, and so chose not to use the Power of Veto. Adria was evicted, and was sent to the jury house.
Season 5 also saw the introduction of a companion talk show on the web. "House Calls" aired weekdays during the entire season and was hosted by former Big Brother 3 houseguest Marcellas Reynolds and radio personality Gretchen Massey. The show featured telephone calls from viewers and interviews with evicted houseguests, houseguests from past seasons and the producers of "Big Brother" Arnold Shapiro and Allison Grodner.
This was also the final season of Big Brother that had the original floorplan of the house that had been built in 2000 used. In the next season, the house was dramatically changed with the addition of another story added on top, with a more luxurious Head of Household room. CBS Corporation, parent company of the network that carries Big Brother, built the new studios of KCBS and KCAL, its two Los Angeles TV stations, on the site; the new studios opened on April 21, 2007.