Reliability and analytical validity of the MX3 hydration testing system during exercise in the heat

Abstract Purpose The MX3 Hydration Testing System provides portable saliva osmolality (SOsm) measurements, but its between-day reliability and analytical validity against a laboratory reference osmometer remain unclear. This study evaluated MX3 reliability and analytical validity and examined whether measurement performance differed between direct tongue and collected-saliva sampling approaches. Methods Twenty healthy, physically active adults (10 females) completed one 30-min familiarization trial and two 120-min self-paced exercise trials in the heat. SOsm was measured using MX3 directly from the tongue and from collected saliva samples and compared with a laboratory freezing-point depression osmometer (OSMO1). Between-day reliability was evaluated using the coefficient of variation (CV), intraclass correlation coefficient (ICC), and Bland–Altman analysis. Analytical validity was assessed using linear regression, CV, and Bland–Altman analysis. Results Direct tongue measurements demonstrated poor between-day reliability (CV = 31–35%; ICC = − 0.003–0.053), whereas collected-saliva measurements demonstrated lower variability and moderate reliability (CV = 17–18%; ICC = 0.618–0.663). Collected-saliva measurements were strongly related to OSMO1 across all measurement conditions (R 2 = 0.570–0.821; all p < 0.001), with mean biases ranging from − 23.9 to − 25.9 mOsm/kg and 95% limits of agreement (LoA) ranging from − 75.4 to 27.1 mOsm/kg. In contrast, direct tongue measurements showed weaker and less consistent relationships with OSMO1 (R 2 = 0.001–0.282; p = 0.005–0.804), greater variability (CV = 36–51%), and larger negative biases (− 31.3 to − 52.0 mOsm/kg), with 95% LoA ranging from − 134.4 to 30.4 mOsm/kg. Agreement between the two MX3 sampling approaches was limited, particularly following exercise. Conclusion Collected-saliva MX3 measurements demonstrated better between-day reliability and analytical performance than direct tongue measurements, although systematic disagreement with the reference osmometer remained. MX3 measurement performance differed between saliva sampling approaches, supporting further validation of the collected-saliva approach.