Low temperature heat release (LTHR) is a phenomenon of interest in both spark ignition (SI) engines and in advanced compression ignition (ACI) engines. Previous studies have demonstrated and investigated LTHR in both ACI and SI engines before the main heat release event and recently, LTHR has been isolated in SI engines by modifying the in-cylinder thermal state during compression and disabling the spark discharge. Nitric Oxide (NO) is a common by-product of combustion in engines. It is formed mostly by oxidation of nitrogen from the air at high temperatures and has been shown to be one of the most reactive residual gas components since it can affect hydrocarbon combustion in small quantities (as low as 30 ppm). In this work, isolated LTHR has been studied under varying levels of NO. Tests were performed on a motored single-cylinder spark ignition engine with inlet air temperatures and pressures adjusted to realise isolated LTHR from mixtures of air, iso-octane (2,2,4-trimethylpentane) and NO. The mixtures were tested with inlet temperatures of 60 °C and 100 °C at an equivalence ratio (ϕ) of 0.5 with boosted (1.5 barA) inlet air conditions. The results show that, contrary to most other published work, the LTHR decreased with increasing NO concentrations for all but one condition tested and NO was shown to inhibit LTHR entirely at higher concentrations. The effect of temperature, pressure, and NO concentration on ignition delay times calculated using a recently developed chemical mechanism are presented alongside experimental pressure-temperature trajectories of the in-cylinder gases to explain the trends. The underlying cause of the trends is explained by using sensitivity analysis to determine the contribution of each reaction within the chemical kinetic mechanism to first-stage ignition, revealing the effect of introducing nitric oxide on the reaction pathways, radical consumption and therefore resulting ignition delay contours and ultimately, LTHR intensity. Novelty and significance statement The effect of nitric oxide (NO) on low temperature heat release (LTHR) has been studied in models but rarely experimentally. Previously, NO was thought to promote LTHR in internal combustion engines. However, this work, looking at the effect of NO on LTHR from iso-octane (2,2,4-Trimethylpentane)-air mixtures at different inlet temperatures, shows that, in general, NO inhibits LTHR. Different chemical kinetics mechanisms are used to explore this behaviour and a recent mechanism from Fang et al. is shown to capture well the behaviour observed experimentally.