Skeletal muscle constantly produces reactive oxygen species (ROS) and nitric oxide (NO) which may play a role in signalling and regulatory pathways. Isolated muscles in vitro release NO to the extracellular space [1] and passive stretching of muscle increases the release of NO from rat skeletal muscle in vitro [2]. We have developed a model to study the generation of ROS and NO in real time in isolated single muscle fibres [3, 4]. The aim of this study was to evaluate the effect of passive stretching on the intracellular generation of ROS and NO in single muscle fibres isolated from young and old wild type and young mdx mice. We used young (2-4 month-old) and old (26-28 month-old) C57BL/6 mice, and young mdx mice, a mouse model of Duchenne muscular dystrophy. Muscle fibres were isolated from the Flexor Digitorus Brevis and attached to a flexible silicone membrane which had been previously coated with a collagen Matrigel™ matrix. Fibres were loaded with different fluorophore probes: 2’, 7’-dichlorodihydrofluorescein-diacetate (DCFH-DA) which is a general detector of ROS, 4-amino-5-methylamino-2’,7’-difluorofluorescein diacetate (DAF-FM-DA) which is a detector of NO, and dihydroethidium (DHE) which can detect superoxide. A passive stretching protocol was applied for eight minutes to fibres using the FX-4000™ Flexercell® system. Using fluorescence microscopy, the fluorescence emission from fibres at different time points was quantified by image analysis to monitor the intracellular ROS and NO. Experimental groups were consisted of 6-12 fibres. Statistical analysis: one-way ANOVA followed by post hoc LSD test for multiple comparisons and unpaired Student’s t test for single comparisons, statistical significance was set at P < 0.05. Results from positive control experiments indicated that the technique is able to detect changes in intracellular ROS and NO. The rate of increase in DAF-FM fluorescence (indicating NO) in fibres from young mice decreased significantly after the application of passive stretching. However, fibres from old mice showed a slight increase in DAF-FM fluorescence after the same stretching protocol, and fibres from young mdx showed no change with the protocol. Fibres from old and young mice displayed different patterns of ethidium fluorescence following DHE loading, with the activity of superoxide appearing to increase in fibres from young mice and decrease in fibres from old mice after the passive stretching protocol. We conclude that the application of passive stretching to isolated single muscle fibres produces minor changes in the intracellular activities of ROS and NO, and induces different patterns of intracellular fluorescence depending on the age of the mice and muscular dystrophy.