Aim: This methodological study investigated the number of brief maximal voluntary isometric contractions (MVC) needed to show evidence of fibre activation, as indicated by changes in the phosphocreatine to creatine (PCr/ Cr) ratio. Methods: Subjects performed series of four, seven and/or 10 MVC (1 s on, 1 s off) of the m. quadriceps (60°-flexion angle). Biopsy samples of the m. vastus lateralis were taken at rest and immediately post-exercise. Single muscle fibres were dissected from the freeze-dried samples and classified as types I, IIA or IIAX, using mATPase stainings. Fragments of characterized fibres were analysed for PCr and Cr content. Analyses of variance were performed to investigate changes in PCr/Cr per fibre group over time, followed by Bonferroni post-hoc test (P < 0.01). The fifth percentile of resting values of each fibre group was determined. Results: Mean PCr/Cr ratio after four, seven and 10 MVCs were significantly lower for all fibre groups (P < 0.01). The mean decreases were 44, 64 and 76%, respectively. However, only after seven and 10 contractions PCr/Cr ratios of all, but three type I and two type IIAX fibres, individual fibres were below the fifth percentile. Conclusion: In very short duration exercise, involving seven brief maximal voluntary contractions, changes in the PCr/Cr ratio indicated activation of different characterized muscle fibre fragments. The results suggest that this approach may be useful for investigating the pattern of fibre type activation in exercise of very short duration.