Musculoskeletal deconditioning is a significant risk during long-term spaceflight. In this study, evaluated the use of a MYOTON probe for spaceflight monitoring of muscular biomarkers. The probe provides data on muscle tone (MT) in Hz, Muscle Stiffness (MS) in N/m, and Muscle Elasticity (ME) via logarithmic decrement. Our initial question was whether use of a guiding belt for the MYOTON probe to measure the paraspinal muscles (PSM) can improve data reliability and accuracy during repeated measurement. The belt significantly reduced standard deviations for ME. Additionally, by comparing our belt data to data on lateral and vertical biomarker deviation, we were able to conclude that use of the guiding belt improves data reliability by measuring at a more appropriate area of the spine. As a second experiment, we wanted to evaluate the extent to which subcutaneous fat thickness can confound MYOTON muscular biomarker data. By applying a quadratic regression model to our data on subcutaneous fat thicknesses at various anatomical loci, we were able to conclude that .689  .035 cm is the maximum fat thickness before MYOTON data become confounded and are no longer valid. Our final experiment aimed to determine if resistance exercise can confound MYOTON data. We found immediate increases in MT and ME following completion of the exercise protocol. All biomarker values returned to baseline within 10 minutes post exercise completion. These experiments help further human spaceflight progress by validating the use of a MYOTON probe for safe and effective monitoring of muscular biomarkers.