Background: Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disease, which causes motoneuron death and has fatal outcome. The clinical picture differs between patients, making the assessment and follow-up much more difficult. Functional ability of such patients is normally assessed using standardized assessment scales and performance tests, while the use of hand-held dynamometry is recommended for assessment of the decrease of the lower limb muscle strength. Sit to stand test can be used to assess functional ability of lower limbs. Purpose: The aim of the study was to evaluate intra-rater reliability of the TRICALS protocols on healthy subjects and to determine the most appropriate number of repetitions to determine the stable outcome of the measurements and to compare results of hand-held dynamometry of lower limbs, five times sit to stand test (5TSTS) and ALSFRS-R scale. Methods: The study included 20 healthy subjects and 30 ALS patients who underwent hand-held dynamometry testing for lower limbs. To assure intra-rater reliability the interclass correlation coefficients (ICC) for the group of healthy participants were calculated. Measurements were performed twice in a span of 5 days. The selection of test results was checked by analysis of variance (ANOVA) and standard measurement error (SEM) was calculated for both groups. To evaluate correlation between the measurements Spearman coefficient was calculated. Results: For determining the most suitable measurement outcome in the use of hand-held dynamometry with the TRICALS protocol, none of the selected values showed statistically significant differences for all tested muscle groups. Compared to the maximum value obtained, the standard measurement error (SEM) values were lower when using an average of three measurements. The intra-rater reliability for hand-held dynamometry in healthy subjects showed a good to high correspondence between the measurements for all muscle groups of the right leg (ICC = 0.87 – 0.97) and moderate to good correspondence for all muscle groups of the left leg (ICC = 0.74 – 0.97). The validity of the muscle strength measurement construct following the TRICALS and 5TSTS protocols did not show statistically significant correlations (ro = -0.16 – -0.56). The correlation between hand-held dynamometry and the functional ALSFRS-R scale was low (ro = 0.001 – 0.28). The correlation between the results of 5TSTS and ALSFRS-R was good (ro = 0.61). Conclusion: The reliability of the intra-rater measurements for muscle strength of the lower limbs in healthy subjects, while following the TRICALS protocol, is high for all muscle groups. For the outcome of hand-held dynamometry, the use of the average value of all measurements seems more reasonable than the use of maximum scores. Moderate to good association was observed in ALS patients between the 5TSTS test and the ALSFRS-R scale, whereas the correlation between hand-held dynamometry and other tests was low. In the future studies it would be reasonable to test the reliability of an individual investigator and reliability among various investigators for hand-held dynamometry following the TRICALS protocol in patients with ALS and to include the 5TSTS test in the clinical patient evaluation, as the assessment of the ability to sit down and stand up is not included in the ALSFRS-R scale.