Athletes are at risk for developing overtraining syndrome, which leads to decrease in athletic performance and increased risk of health problems (for example overuse injuries). Diagnosing the syndrome is complicated, and there is currently no good diagnostic marker. One potential marker is a decrease in the testosterone to cortisol ratio (T/C ratio) compared to the athlete's basal value, but the methodology for using different forms of testosterone is not uniform across the literature. The main objective of this work was to investigate a correlation between the change in the T/C ratio and the probability of the presence of overtraining syndrome, using different forms of testosterone to calculate the ratio. We hypothesised that calculated free and bioavailable testosterone in the T/C ratio would be better markers of increased likelihood of overtraining syndrome than total testosterone. As part of the master's thesis, 93 elite athletes of the 1st Slovenian Handball League were observed and venous blood samples were taken from them during the course of one handball season. In serum samples testosterone, cortisol and SHBG were measured by the electrochemiluminescent method, and albumin was determined by the colorimetric method using bromcresol green. The Vermeulen equation was used to calculate free and bioavailable testosterone. The T/C ratio was calculated using the measured total testosterone and the calculated forms The baseline T/C ratio was based on the results of the sampling performed before the training season. Two additional samplings were carried out during the season. We found statistically significant differences (p=0,03) in the change in T/C ratio from baseline between the groups with lower and higher probability of overtraining syndrome, when total testosterone is used to calculate the ratio. When using free (p=0,07) or bioavailable testosterone (p=0,052) to calculate the ratio, we did not detect statistically significant differences. The results of the logistic regression showed that a decrease in the T/C ratio using total testosterone increases the chance of overtraining syndrome (p=0,027). The T/C ratio threshold was determined using the receiver operating characteristic (ROC) curve and ranges from a 29-31% reduction in the T/C ratio, depending on the form of testosterone used. The results of the statistical analysis indicate that total testosterone used in the T/C ratio is a better marker of probability of overtraining syndrome than free and bioavailable testosterone.
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