Testosterone and Cortisol Salivary Samples Are Stable Across Multiple Freeze-Thaw Cycles

Sontag, SA, Cabarkapa, D, and Fry, AC. Testosterone and cortisol salivary samples are stable across multiple freeze-thaw cycles. J Strength Cond Res 37(4): 915-918, 2023-When processing salivary samples for biomarker analysis, avoiding multiple freeze-thaw cycles is generally recommended. However, confusing tissue handling instructions or challenges with collections in the field sometimes makes this problematic. Thus, the purpose of this study was to examine if the stability of salivary testosterone (T) and cortisol (C) hormones remains unchanged when exposed to multiple freeze-thaw cycles. Seven healthy recreationally active adults provided salivary samples at rest (i.e., 1600 hours) for analysis of T and C. Samples were separated into 4 aliquots for each hormone and underwent 4 freeze-thaw cycles (T1-T4 and C1-C4) before being analyzed by enzyme-linked immunosorbent assay. The overall analysis of variance model was significant for T ( p = 0.008) and nonsignificant for C ( p = 0.820). A follow-up post hoc comparison indicated significant differences in salivary hormonal concentrations between T1 and T4 ( p = 0.029), T2 and T4 ( p = 0.007), and T3 and T4 ( p = 0.032). The findings of this study indicate that salivary steroid hormones seem to be relatively stable following multiple freeze-thaw cycles. However, C seems to be more stable when exposed to multiple freeze-thaw cycles, as T concentrations did reveal a significant decrease by the fourth thaw cycle.