Gain-of-function RyR1-p.R163C mutation in ryanodine receptors type 1 (RyR1) deregulates Ca signaling and mitochondrial function in skeletal muscle and causes malignant hyperthermia in humans and mice under triggering conditions. We investigated whether T lymphocytes from heterozygous RyR1-p.R163C knock-in mutant mice (HET T cells) display measurable aberrations in resting cytosolic Ca concentration ([Ca]), Ca release from the store, store-operated Ca entry (SOCE), and mitochondrial inner membrane potential (ΔΨ) compared with T lymphocytes from wild-type mice (WT T cells). We explored whether these variables can be used to distinguish between T cells with normal and altered RyR1 genotype. HET and WT T cells were isolated from spleen and lymph nodes and activated in vitro using phytohemagglutinin P. [Ca] and ΔΨ dynamics were examined using Fura 2 and tetramethylrhodamine methyl ester fluorescent dyes, respectively. Activated HET T cells displayed elevated resting [Ca], diminished responses to Ca mobilization with thapsigargin, and decreased rate of [Ca] elevation in response to SOCE compared with WT T cells. Pretreatment of HET T cells with ryanodine or dantrolene sodium reduced disparities in the resting [Ca] and ability of thapsigargin to mobilize Ca between HET and WT T cells. While SOCE elicited dissipation of the ΔΨ in WT T cells, it produced ΔΨ hyperpolarization in HET T cells. When used as the classification variable, the amplitude of thapsigargin-induced Ca transient showed the best promise in predicting the presence of RyR1-p.R163C mutation. Other significant variables identified by machine learning analysis were the ratio of resting cytosolic Ca level to the amplitude of thapsigargin-induced Ca transient and an integral of changes in ΔΨ in response to SOCE. Our study demonstrated that gain-of-function mutation in RyR1 significantly affects Ca signaling and mitochondrial fiction in T lymphocytes, which suggests that this mutation may cause altered immune responses in its carrier. Our data link the RyR1-p.R163C mutation, which causes inherited skeletal muscle diseases, to deregulation of Ca signaling and mitochondrial function in immune T cells and establish proof-of-principle for in vitro T cell-based diagnostic assay for hereditary RyR1 hyperfunction.