Eufemiano Fuentes said the vast majority of those who approached him were cyclists, but others also came to him. He did not give any names.
"There were sports people of all sorts," Fuentes said.
Pressed by state prosecutor Rosa Calero, Fuentes added: "They could have been a cyclist from a given team, a footballer from a squad, a tennis player, I don't know, a boxer. There was an athlete."
The doctor said he was approached to perform medical checks and help prepare training and diet regimens.
"I did physical and medical tests to guarantee their health was not harmed by the rigours of competition," Fuentes said.
Fuentes said this involved blood doping, with red blood cells extracted from a person and later reinjected to boost stamina and performance.
Fuentes insisted the transfusions never took place during competitive races, except when a rider retired and needed an instant boost of red cells to avoid immediate health risks.
Fuentes said the majority of transfusions took place in what he called a medical centre in downtown Madrid. However, he conceded under cross-examination that some took place in nearby hotel rooms, when sporting competitors asked for greater privacy and discretion.
Calero and Lucia Pedreno, a lawyer representing the Spanish state, spent more than four hours cross-examining Fuentes about the high technology centrifuges and refrigeration equipment he used and the notes he used to keep track of the hundreds of blood bags.
Fuentes said that sometimes he used chilled soft drinks cans to keep blood bags cool when he transported them over short distances in insulated carrying containers.
During the trial, Fuentes declined to answer a number of questions, including ones about how he disposed of unwanted blood bags and some referring to privacy agreements between himself and his clients.
He used a legal right similar to the Fifth Amendment which enables defendants to avoid incriminating themselves.
Also Tuesday, presiding judge Julia Santamaria agreed with all the parties that American cyclist Tyler Hamilton would be called to testify as a witness. Hamilton has been outspoken in revealing details of doping in the sport.
Hamilton was a former teammate of Lance Armstrong. His tell-all interview on "60 Minutes" in 2011, combined with his testimony and a book he wrote last year, played a key part in Armstrong's downfall.
After being stripped of his seven Tour de France titles and banned from the sport for life, Armstrong has now admitted that he doped.
Santamaria said she would consider written applications from prosecution and plaintiffs about what to do with scores of blood bags seized by police but not used as evidence in the Puerto case.
She said the trial would not make a distinction between medical drugs used for doping purposes and blood products used for the same reason.
The trial would only focus on whether the defendants posed a risk to public health in the services they offered sports men and women.
Thirty-five witnesses are expected to testify in a trial due to last until March 22.
Although no riders are on trial, many will be called to testify as witnesses, including two-time Tour de France champion Alberto Contador. He was stripped of a third Tour title after testing positive for clenbuterol.
The trial is limited to doping in cycling, even though athletes in other sports were also reportedly implicated in the discovery of blood bags and other doping equipment in 2006. The World Anti-Doping Agency wants all evidence to be released.
Santamaria can rule only on matters covered by Spanish law as it applied in May 2006, when police raids uncovered a mass of blood doping evidence in labs, offices and apartments.
Also on trial are Fuentes' sister and fellow doctor, Yolanda; Manolo Saiz, a former ONCE and Liberty Seguros team sports director; and Vicente Belda and Ignacio Labarta, both associated with the former Kelme team.
Jose Luis Merino, another doctor, also was due to be tried, but Santamaria granted him a temporary stay last week after he presented medical reports stating he is suffering from Alzheimer's.