Cain stayed in the game in the outfield for another half-inning Wednesday before realizing he was injured.
"I tried not to show it [running back to the dugout]," he said. "I definitely felt it. I was hoping it wasn't anything. When I went back to the outfield and tried to run, I knew something was wrong. I let the trainers know."
Jarrod Dyson replaced Cain, singling and scoring a run in the 11th inning of the 6-4 Kansas City victory. Dyson started Thursday night's game vs. the Astros in center field.
The Royals called up left-hander Justin Marks from Triple-A Omaha in time for Thursday night's game at Houston to replace Cain on the active roster.
In 2012, Cain was limited to 61 games by three different leg injuries which included a left groin strain as well as a torn left hip flexor and a right hamstring strain. Last year he spent 15 days on the disabled list with a pulled oblique.
"It's frustrating," he said. "I've been working hard to keep my leg healthy. That's my main focus this year, stay healthy, stay on the field. I have to stay positive. Keep rooting on my teammates. Hope they keep winning and when I get back, we'll continue to win.
"You wait a few days, let it heal, then start the process of stretching it. I'm definitely used to it, that DL process. Once I get back on the field, I'll be ready to go."
Manager Ned Yost said that when the Royals return to Kansas City, opening a weekend series against Minnesota, the team doctors will be able to examine Cain and give him an MRI.
"[Trainer Nick Kenney] thinks it can be 5-7 days, best case scenario," Yost said. "He isn't any better [today] than he was yesterday. Still sore in his leg. We'll give him time to get this out of the way. Hopefully it won't be more than 15 days, but we'll see."
Marks is 1-1 with a 3.24 ERA in three games for Omaha, including one start.
Cain injured himself trying to beat out a ground ball to shortstop in the ninth inning with the game tied. First-base umpire CB Bucknor called Cain safe, but Houston manager Bo Porter challenged the call and replays proved Cain was out.
"I thought I was safe," Cain said. "But they overturned it. It happens. And I get hurt on top of it. I'm sure I'll get some overturned and win a few. It's a little frustrating. That's why we've got [replay]. If they got it right, they got it right. We still won. That's all that counts. Another win for the good guys."
Yost expressed confidence that Dyson can do the job in center field.
"I'm real confident," Yost said. "He's done a really good job of identifying the type of player he is. He had more walks than strikeouts in Spring Training. We know when he gets on, it takes almost a miracle to throw him out."