Introduction A healthy diet and high health-related physical fitness levels may be part of an overall healthy lifestyle. The relationship between adherence to the Mediterranean diet and physical fitness levels has been analyzed in several studies. However, no studies have synthesized evidence on this relationship throughout adulthood. Moreover, in addition to the overall Mediterranean dietary pattern, the associations of individual components of the Mediterranean diet with physical fitness indicators are also unclear. Methods This protocol for a systematic review and meta-analysis was conducted according to the Preferred Reporting Items for Systematic Review and Meta-Analysis for Protocols statement and the Cochrane Collaboration Handbook. Systematic literature searches will be performed in the MEDLINE (PubMed), Scopus, Web of Science, SPORTDiscus and Cochrane CENTRAL databases to identify studies published up to 31 January 2022. The inclusion criteria will comprise observational studies and randomized controlled trials reporting the associations between adherence to the Mediterranean diet and physical fitness levels on general healthy or unhealthy adults (≥18 years). When at least five studies addressing the same outcome are available, meta-analysis will be carried out to estimate the standardized mean difference of physical fitness according to the adherence to Mediterranean diet. Subgroup analyses will be performed according to the characteristics of the population, the individual dietary components of the Mediterranean diet and physical fitness parameters as long as there are sufficient studies. Ethics and dissemination This systematic review and meta-analysis protocol is designed for updating evidence on the associations between adherence to overall Mediterranean diet (and specific Mediterranean foods) and physical fitness levels in young, middle-aged, and older adults. Findings from this review may have implications for public health. The results will be disseminated through peer-reviewed publication, conference presentation, and infographics. No ethical approval will be required since only published data will be used. PROSPERO registration number CRD42022308259.