Prior to drinking the water from the Fountain of Youth, the narrator cites that Colonel Killigrew "wasted his best years." In his youth, he had been promiscuous and as an older man, he ended up with gout and other painful diseases and ailments.
He, Mr. Medbourne, and Mr. Gascoigne were once lovers of Widow Wycherly. Given that he preferred to be with many women rather than just one, he may have missed an opportunity to be with only the Widow herself.
Later in the story, the Widow alludes to the fact that the Colonel's "compliments were not always measured by sober truth." If he was in fact often drunk, it is likely he lost or missed other opportunities as a result of this.
As for what he lost or wasted after drinking the water, we can conclude that he, Mr. Redbourne, Mr. Gascoigne, and Widow Wycherly all missed the chance to learn a harsh lesson. Dr. Heidegger sees that a taste of youth only makes them want it more. It is a thirst that can not be quenched and therefore, probably should not be indulged at all. The colonel had been lecherous as a younger man, feeding his insatiable desires. By the end of the story, he has simply traded one insatiable desire for another. He has wasted an opportunity to learn from his mistakes, as have the other three involved in Dr. Heidegger's experiment.