At the 1960 Summer Olympics in Rome, Italy, Rudolph competed in three events on a cinder track in Rome's Stadio Olimpico: the 100- and 200-meter sprints, as well as the 4 × 100-meter relay. Rudolph, who won a gold medal in each of these events, became the first American woman to win three gold medals in a single Olympiad.

Rudolph ran the finals in the 100-meter dash in a wind-aided time of 11.0 seconds. (The record-setting time was not credited as a world record, because the wind, at 2.75 metres (3.01 yd) per second, exceeded the maximum of 2 metres (2.2 yds).) Rudolph became the first American woman to win a gold medal in the 100-meter race since Helen Stephens's win in the 1936 Summer Olympics. Rudolph won another gold medal in the finals of the 200-meter dash with a time of 24.0 seconds, after setting a new Olympic record of 23.2 seconds in the opening heat. After these wins she was hailed throughout the world as "the fastest woman in history."

On September 7, 1960, with thousands of spectators jammed the stadium. Rudolph combined efforts with her Olympic teammates from Tennessee State (Martha Hudson, Lucinda Williams, and Barbara Jones) to win the 4 × 100-meter relay with a time of 44.5 seconds, after setting a world record of 44.4 seconds in the semifinals. Rudolph ran the anchor leg for the American team in the finals and nearly dropped the baton after a pass from Williams, but she overtook Germany's anchor leg runner to win the relay in a close finish.

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