Introduction It is usual in physiological research to assess the effects of some intervention on extremities (e.g., training programmes or injury recovery protocols) using one muscle for the intervention and its contralateral as control. For methodological and ethical reasons, the laboratory rat is used as animal model and an obvious question, which has been posed few times in the scientific literature, arises: could it be any structural, metabolic or functional difference between rat right and left muscles? In rats, gastrocnemius is one of the muscles most widely used in research because of its importance in locomotion and high relative leg mass. To the best of our knowledge, only the study by Guo and Zhou (2003) reported a laterality assessment on the metabolic profile of rat’s gastrocnemius muscle. To deepen the knowledge on laterality in rats and to clarify the contralateral muscles interchangeability for experimental design, our study aimed to evaluate the force contractile properties between right and left rat’s gastrocnemius muscle. Material and methods Muscle force properties were analysed in right and left gastrocnemius muscles from six male rats using ADInstruments (Oxford, UK) hardware and software. Gastrocnemius was isolated from surrounding tissues, the Achilles tendon attached to a force transducer (MLT 1030/D) and the muscle stimulated (Stimulus Isolator FE180) via its sciatic nerve. The following force parameters were registered with data acquisition hardware PowerLab/16SP and analysed with LabChart v7.3.7 software: peak (PF, mN/g) and tetanic forces (TF, mN/g) and contraction (CT) and half-relaxation times (HRT) in milliseconds (ms). Fatigue properties after low frequency (40 Hz) continuous stimulation were also assessed as a fatigue index (FI) that considered the force-time area to baseline after 2 min. At the end of the experiment, both gastrocnemius were excised and weighed to the nearest 0.01 g. Protocols were performed following the European Union and Spanish Law on Animal Protection and approved by the Local Bioethics Committee. Data were statistically analysed running paired t-Student tests (SigmaPlot 11, Systat Software, San Jose, US) and values expressed as mean ± standard deviation. Results No statistically significant differences (P=0.266) were found in gastrocnemius to body weight ratio (‰) between right (6.31±0.32) and left (6.41±0.27) muscles. Force parameters normalized to gastrocnemius weight (in mN·g-1) did not show any significant difference between right (PF=74.0±13.4, TF=219.4±13.0) and left (PF=70.9±10.7, TF=213.0±18.0) legs with P=0.623 (PF) and P=0.514 (TF). Twitch time parameters (in ms) lacked also of significant differences between legs (CT: 43.4±8.6 vs 45.0±14.3, P=0.639; HRT: 77.6±15.0 vs 82.3±25.3, P=0.475). Finally, both muscles also showed similar (P=0.718) fatigue properties (in N·s) with FI=16.5±1.3 (right) and FI=16.1±2.0 (left). Conclusion Strength, muscle mass and fatigue and force contractile properties did not statistically differ between right and left gastrocnemius in rats. This absence of laterality at the functional level raises the possibility of using right and left gastrocnemius interchangeably for experimental designs were one muscle is used to analyse data after a physiological intervention and its contralateral muscle plays the control role, thus allowing unbiassed paired comparisons to derive accurate conclusions.