Background: Mitochondrial diseases (MDs) are a group of genetic disorders with diverse patient phenotypes resulting from mitochondrial dysfunction. Pathologies are often poorly understood, and therapies are limited. Previously, we identified abnormalities in glutamine metabolism as well as an upregulation of the integrated stress response (ISR) and mitochondrial unfolded protein response (UPRmt) in dermal fibroblasts of patients with the dilated cardiomyopathy with ataxia syndrome (DCMA), a systemic mitochondrial disease (MD) caused by deficiency of DNAJC19. However, it is unclear whether these changes are unique to DCMA or shared across other MDs. Aims: To investigate changes in glutamine metabolism and stress responses in dermal fibroblasts from patients with Barth Syndrome (BTHS), complex I deficiency (C1D), and Kearns-Sayre syndrome (KSS) and healthy fibroblasts treated with mitochondrial disruptors. We hypothesized that, regardless of cause, mitochondrial dysfunction will lead to abnormal glutamine metabolism and activation of the ISR and UPRmt. Methods: Fibroblasts from healthy individuals (N=6) and patients with a diagnosis of BTHS (N=2), C1D (N=3) and KSS (N=3) were cultured in media containing heavy isotope-labelled glutamine [5-13C 2-15N]. After 48 hours, extracellular media was extracted and metabolites were quantified using liquid-chromatography-mass spectrometry (LC-MS). To validate findings, we also treated healthy fibroblasts (N=2) with dinitrophenol (DNP) and rotenone. The expression of ISR- and UPRmt-associated genes was quantified via qPCR. Results: Levels of the dual-heavy isotope-labelled glutamine (5-13C 2-15N) and glutamine-derived isotopomers were not significantly different in C1D, KSS and BTHS, compared to controls. In healthy fibroblasts, DNP (1 mM) reduced 5-13C 2-15N glutamine uptake, whereas rotenone (100 nM) had no effect. The ISR and UPRmt were not upregulated in BTHS, C1D, or KSS although data were limited by small numbers. Exposure to 500 nM rotenone and 1 mM DNP significantly upregulated ISR/UPRmt-associated genes ATF4 and ATF5 in healthy fibroblasts. Conclusions: These preliminary findings suggest that glutamine metabolism and the activation of cellular stress responses in MD fibroblasts differs depending on extent and/or etiology of mitochondrial dysfunction. Our results support the observation that DCMA fibroblasts have a unique dysregulation of glutamine metabolism and stress responses which may have implications for pathophysiology and treatment.