*2.4. Physical Exercise Oxidative Stress Model*

The model was a high-intensity and long-lasting physical activity (90 min) on a bicycle roller. Participants underwent a preliminary test and two subsequent tests (test #1 after a 7-day washout period and test #2 at the end of the study at 6 weeks). The preliminary test was performed to calculate the intensity of tests #1 and #2 for each individual, using a bicycle roller with electromagnetic resistance (Technogym Spin Trainer) with an initial speed load of 12 km/h, with a 2 km/h load increase every minute, maintaining a constant slope of 2%. The cyclists employed free development. In order to calculate the intensity of tests #1 and #2, participants were monitored by ECG and gas analyzer (Jaeger Oxicom Pro®, CareFusion Respiratory Care, Germany) to determine maximal heart rate (MHR) and monitor heart rate above anaerobic threshold and during maximum oxygen uptake (VO2 max). Tests #1 and #2 lasted 90 min, and the maximum maintained load was equivalent to a heart rate corresponding to 75% of VO2 max calculated in the preliminary test. A constant slope of 2% was also used. The water consumption was ad libitum. After test #1, participants were given the assigned supplement (probiotic or placebo). Forty-eight hours before each test participants did not make any intense physical or psychological effort.

#### *2.5. Study Procedures*

The study included three visits, one at baseline during the time of the preliminary test, one at the time of test #1, and a final visit after test #2 at 6 weeks. At baseline, participants signed the informed consent, when eligibility criteria were checked, and the study product was given. Clinical evaluations included detailed medical history and measurement of anthropometric variables. Compliance with the intake of the probiotic product was assessed by counting the remaining capsules in the medication container. Adverse events were ascertained by directly asking participants how they were feeling after taking the product and from abnormal changes of laboratory results. During the study period, there were no dietary restrictions, but medications that may affect the microbiome (e.g., antioxidants, statins) were not allowed.

Peripheral blood samples (12 mL) after 12 h fasting were extracted at 30 min before and after each test, and 24 h urine samples were collected one day before and after the test. From the total urine volume, a 9 mL sample was frozen at −80 ◦C for further analysis.

Stool samples were collected during 24 h before test #1 and at 6 weeks during the 24 h before test #2, preserved with REAL stock buffer (Durviz S.L., Paterna, Valencia, Spain), and stored at −80 ◦C until analysis.
