The purpose of this study was to compare the reliability of trunk muscle activity measured by means of surface electromyography (EMG) during maximal and sub-maximal voluntary isometric contractions (MVC/sub-MVC) over repeated trials within-day and between-days in healthy controls and patients with chronic low back pain (CLBP). Eleven volunteers (six controls and five CLBP patients) were assessed twice with a 1-week interval. Surface EMG signals were recorded bilaterally from six trunk muscles. Intra-class correlation coefficients (ICC) and standard error of measurement as a percentage of the grand mean (%SEM) were calculated. MVC and sub-MVC showed excellent within-day reliability in both healthy controls and CLBP patients (ICC mean 0.91; range 0.75-0.98; %SEM mean 4%; range 1-12%). Sub-MVC for both groups between-days showed excellent reliability (ICC mean 0.88; range 0.78-0.97; %SEM mean 7%; range 3-11%). The between-days MVC for both groups showed trends towards lower levels of reliability (ICC mean 0.70; range 0.19-0.99; %SEM mean 17%; range 4-36%) when compared to sub-MVC. Findings of the study provide evidence that sub-MVC are preferable for amplitude normalisation when assessing EMG signals of trunk muscles between-days.