Quantifying sweat gland activation provides important information when explaining differences in sweat rate between populations and physiological conditions. However, no standard technique has been proposed to measure sweat gland activation, while the reliability of sweat gland activation measurements is unknown. We examined the interrater and internal reliability of the modified-iodine paper technique, as well as compared computer-aided analysis to manual counts of sweat gland activation. Iodine-impregnated paper was pressed against the skin of 35 participants in whom sweating was elicited by exercise in the heat or infusion of methylcholine. The number of active glands was subsequently determined by computer-aided analysis. In total, 382 measurements were used to evaluate: 1) agreement between computer analysis and manual counts; 2) the interrater reliability of computer analysis between independent investigators; and 3) the internal reliability of sweat gland activation measurements between duplicate samples. The number of glands identified with computer analysis did not differ from manual counts (68 ± 29 vs. 72 ± 24 glands/cm(2); P = 0.27). These measures were highly correlated (r = 0.77) with a mean bias ± limits of agreement of -4 ± 38 glands/cm(2). When comparing computer analysis measures between investigators, values were highly correlated (r = 0.95; P < 0.001) and the mean bias ± limits of agreement was 4 ± 18 glands/cm(2). Finally, duplicate measures of sweat gland activation were highly correlated (r = 0.88; P < 0.001) with a mean bias ± limits of agreement of 3 ± 29 glands/cm(2). These results favor the use of the modified-iodine paper technique with computer-aided analysis as a standard technique to reliably evaluate the number of active sweat glands.