Specific force (SF) represents a muscle’s contractile quality and is the peak isometric force normalised to cross-sectional area (CSA). There is large variance in published SF measurements from human skinned muscle fibres of young, healthy individuals, reduced only in part when methodological differences between studies are accounted for (Kalakoutis et al., 2014). Despite research on the effects of different chemical substrates on the mechanical properties of skinned fibres, experimental solutions used differ between research groups. The aim was to quantify the effect of this methodological difference, with emphasis on SF production. Human Vastus Lateralis muscle fibres (n = 96) were obtained from a biopsy sample following local anaesthesia (2% lidocaine) in one young, healthy, male. Fibres were chemically skinned and exposed to two different activating solutions, A and B, in a random order. SF was measured at 15°C and time to half peak tension (t50) was calculated as an indication of contraction kinetics. Three differences between solutions A and B were the uses of: 1) Imidazole (A) or Tes (B) 2) Glutathione (GL) (B only) 3) Potassium Chloride (KCl) (A) or Potassium Propionate (K-prop) (B). The impact of each difference on skinned fibre contraction was isolated by making new solutions which differed in only one chemical constituent. A paired t-test assessed significance (p<0.05) of mechanical results (mean ± SD). 1H nuclear magnetic resonance spectroscopy (1H NMR) monitored potential formation of new compounds by reaction of Imidazole or Tes, which could affect SF. A significantly higher SF and shorter t50 was measured from the same fibres in solution B (109.8 ± 45.3 kPa; 1.5 ± 0.9s) compared with solution A (75.8 ± 43 kPa; 9.4 ± 4.4s). Isolating the effects of individual chemical components showed SF was 15.4 ± 7.7% higher (p<0.05) in a solution containing an optimum concentration of Tes (60mM), not Imidazole (20mM). The t50 was shorter (p<0.05) in solution containing GL (2.3 ± 1.1s) compared to without GL (5.8±2.9s) and in a solution containing K-prop (1.5 ± 1.4s) compared with a similar solution containing KCl (4.0 ± 3.0s). 1H NMR spectra corresponded to the compounds expected based on each solution’s composition, with no indication of reaction products. The higher SF elicited in solution B was largely due to the use of Tes instead of Imidazole. The shorter t50 in solution B was partly accounted for by a lower Cl- concentration due to the use of K-prop instead of KCl and by the use of GL. 1H NMR experiments could not measure effects on ionic strength or [Mg2+] so these remain possible mechanisms of the higher SF elicited by solution B. These findings show that the use of different experimental solutions contributes substantially to the disparity of SF measurements reported by different publications studying human skinned fibres.