Herein, we present a study focused on the determination of the influence of long-distance (53 km) bicycle riding on levels of chosen biochemical urinary and serum prostate cancer (PCa) biomarkers total prostate-specific antigen (tPSA), free PSA (fPSA) and sarcosine. Fourteen healthy participants with no evidence of prostate diseases, in the age range from 49–57 years with a median of 52 years, underwent physical exercise (mean race time of 150 ± 20 min, elevation increase of 472 m) and pre- and post-ride blood/urine sampling. It was found that bicycle riding resulted in elevated serum uric acid (p
= 0.001, median 271.76 vs.
308.44 µmol/L pre- and post-ride, respectively), lactate (p
= 0.01, median 2.98 vs.
4.8 mmol/L) and C-reactive protein (p
= 0.01, 0.0–0.01 mg/L). It is noteworthy that our work supports the studies demonstrating an increased PSA after mechanical manipulation of the prostate. The subjects exhibited either significantly higher post-ride tPSA (p
= 0.002, median 0.69 vs.
1.1 ng/mL pre- and post-ride, respectively) and fPSA (p
= 0.028, median 0.25 vs.
0.35 ng/mL). Contrary to that, sarcosine levels were not significantly affected by physical exercise (p
= 0.20, median 1.64 vs.
1.92 µmol/mL for serum sarcosine, and p
= 0.15, median 0.02 µmol/mmol of creatinine vs.
0.01 µmol/mmol of creatinine for urinary sarcosine). Taken together, our pilot study provides the first evidence that the potential biomarker of PCa—sarcosine does not have a drawback by means of a bicycle riding-induced false positivity, as was shown in the case of PSA.
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