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A New Game + is the most common reference to the second playthrough of No More Heroes. It can be accessed after defeating Henry in the game's "real" ending and saving the clear data to another data slot. From there the player will be returned to the very beginning of the game, although this second playthrough offers a number of features that are exclusive to a New Game +. It can also be accessed by pausing the game, opening the Data screen, selecting Load Data, and loading the slot where the clear data was saved to.

From the beginning of a New Game +, the player will retain all of their acquired trading cards, clothing, beam katanas and beam katana parts, Beam Katana Combo, Strength and Maximum vitality stats and techniques. Any missions, assassination gigs and free fights that were available to the player, as well as the resulting ranks that were earned, will also be retained during a New Game +. Also, if the player chooses, the game can be replayed at a different difficulty level (including hard, or "Bitter" mode which is unlocked after the game's first initial playthrough); playing at a different difficulty level is not required however, but is recommended so that enemies can "keep up" with the weapons, stats and techniques the player has accumulated during their first playthrough.

Starting a New Game + will also add one hundred new trading cards to be collected by the player. This "Design Materials" set features cards containing different types of concept art from the game, including character designs, expressions, weapons, profile cards, and art of several other things surrounding the game, including Pure White Lover Bizarre Jelly. Not unlike the first playthrough, many of these new trading cards are obtained from chests, though the majority are found by unearthing them from particular sections of Santa Destroy where they are now buried. As for uncovering these buried trading cards, if the player has purchased the Accelerator from Naomi's Lab, when in the vicinity of buried treasure the Wii Remote will make a slight vibration. If the player progresses in the right direction towards the buried treasure's location, the Wii Remote will continue to make brief vibrations. If the player progresses in the wrong direction towards the buried treasure, the Wii Remote will no longer make these vibrations. As the player progresses closer to the buried treasure, the vibrations will intensify, and when the player is standing over, or extremely close to the treasure, the Wii Remote will make constant, intense vibrations. If the player has not obtained the Accelerator, this can be a difficult process of trial and error.

Also, starting a New Game + will cause the t-shirt selection at Area 51 to expand.

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