The Los Angeles Angels ace, who led Japan to victory in the World Baseball Classic last month, fell foul of baseball's new time violation rules during his team's 4-3 road win over the Seattle Mariners.
Ohtani, making his second start of the season from the mound, picked up the first clock violation at the top of the first inning after being adjudged to have begun his delivery before the hitter was ready.
That led to home-plate umpire Pat Hoberg giving a free ball to Mariners catcher Cal Raleigh (26).
Ohtani was then penalised again in the top of the sixth inning as he stepped to the plate to face Mariners reliever Matt Brash (24).
Hoberg ruled Ohtani had taken too long to step into the batter's box, meaning the Angels slugger was hit with an automatic strike.
The pitch clock was introduced by Major League Baseball this year as part of efforts to speed up play in the sport.
Pitchers have a 15-second clock when the bases are empty with batters having to be ready to hit with eight seconds remaining.
Hurlers get 20 seconds with runners on base and 30 seconds between hitters.
So far the innovation has been largely successful, with pre-season games shaving around 25 minutes off the length of a typical baseball game.
