The current study investigated the reliability and validity of a novel non-motorised treadmill(NMT) based soccer simulation utilising a novel activity category called a ‘variable run’to quantify fatigue during high-speed running. Twelve male University soccer players completed three familiarisation sessions and one peak speed assessment before completing the Intermittent Soccer Performance Test (iSPT) twice. The two iSPT’s were separated by 6 –10 days. The total distance, sprint distance and high-speed running distance were 8968 ± 430m, 980 ± 75 m and 2122 ± 140 m, respectively. No significant difference (p>0.05) was found between repeated trials of the iSPT for all physiological and performance variables. Reliability measures between iSPT1 and iSPT2 showed good agreement (CV: <4.6%; ICC: >0.80). Furthermore, the variable run phase showed high-speed running distance significantly decreased (p<0.05) in the last 15 min (89 ± 6 m) compared to the first 15 min (85 ± 7 m), quantifying decrements in high-speed exercise compared to previous literature. The current study validates the iSPT as a NMT based soccer simulation compared to previous match-play data, and is a reliable tool for assessing and monitoring physiological and performance variables in soccer players. The iSPT could be utilised in a number of ways including player rehabilitation, understanding the efficacy of nutritional interventions, and also the quantification of environmentally mediated decrements upon soccer-specific performance.