We used the nanometer-wide tubules of the transverse tubular (t)-system of human skeletal muscle fibers as sensitive sensors for the quantitative monitoring of the Ca²⁺-handling properties in the narrow junctional cytoplasmic space sandwiched between the tubular membrane and the sarcoplasmic reticulum cisternae in single muscle fibers. The t-system sealed with a Ca²⁺-sensitive dye trapped in it is sensitive to changes in ryanodine receptor (RyR) Ca²⁺ leak, the store operated calcium entry flux, plasma membrane Ca pump, and sodium–calcium exchanger activities, thus making the sealed t-system a nanodomain Ca²⁺ sensor of Ca²⁺ dynamics in the junctional space. The sensor was used to assess the basal Ca²⁺-handling properties of human muscle fibers obtained by needle biopsy from control subjects and from people with a malignant hyperthermia (MH) causative RyR variant. Using this approach we show that the muscle fibers from MH-susceptible individuals display leakier RyRs and a greater capacity to extrude Ca²⁺ across the t-system membrane compared with fibers from controls. This study provides a quantitative way to assess the effect of RyR variants on junctional membrane Ca²⁺ handling under defined ionic conditions.