Duchenne muscular dystrophy (DMD), an X-linked childhood-onset muscular dystrophy caused by loss of the protein dystrophin, can be associated with neurodevelopmental, emotional and behavioural problems. A DMD mouse model also displays a neuropsychiatric phenotype, including increased startle responses to threat which normalise when dystrophin is restored in the brain. We hypothesised that startle responses may also be increased in humans with DMD, which would have potential translational therapeutic implications. To investigate this, we first designed a novel discrimination fear-conditioning task and tested it in six healthy volunteers, followed by male DMD (n = 11) and Control (n = 9) participants aged 7-12 years. The aims of this methodological task development study were to: i) confirm the task efficacy; ii) optimise data processing procedures; iii) determine the most appropriate outcome measures. In the task, two neutral visual stimuli were presented: one 'safe' cue presented alone; one 'threat' cue paired with a threat stimulus (aversive noise) to enable conditioning of physiological startle responses (skin conductance response, SCR, and heart rate). Outcomes were the unconditioned physiological startle responses to the initial threat, and retention of conditioned responses in the absence of the threat stimulus. We present the protocol development and optimisation of data processing methods based on empirical data. We found that the task was effective in producing significantly higher physiological startle SCR in reinforced 'threat' trials compared to 'safe' trials (P < .001). Different data extraction methods were compared and optimised, and the optimal sampling window was derived empirically. SCR amplitude was the most effective physiological outcome measure when compared to SCR area and change in heart rate, with the best profile on data processing, the least variance, successful conditioned response retention (P = .01) and reliability assessment in test-retest analysis (rho = .86). The definition of this novel outcome will allow us to study this response in a DMD population.