Game:

Shinobi III: Return of the Ninja Master (1993)

Duration: 1:06:43

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This is the hardest difficulty available in the game. It features two pretty crucial differences:
. Your maximum health is reduced by half.
. You don't get any extra lives from score.

I don't know if there are other differences, but those seem to be the only relevant ones. Essentially, you just need to play the game more carefully to make less mistakes.
Normally I don't bother with higher difficulties in games like this one, but I like the artistic side of this game so much, that I felt like it deserved it. The first five stages are also pretty fun to play too.