Reply - Letter to the editor - ‘Beetroot juice intake positively influenced gut microbiota and inflammation but failed to improve functional outcomes in adults with Long COVID’

This reply addresses concerns regarding the analytical approach used in a pilot trial investigating beetroot juice intake in adults with Long COVID. The critique focused on potential biases inherent to partial least squares–discriminant analysis (PLS-DA) and associated feature importance metrics. To ensure reliability, the study incorporated repeated double cross-validation and permutation testing, widely regarded as effective strategies to minimize overfitting and spurious feature selection. Multiple feature importance measures and comparative analyses, including Spearman’s rank correlations, were applied to confirm the stability and biological relevance of identified markers. The convergence of results across validation methods supports the robustness of the analytical strategy and strengthens confidence in the reported findings. This response highlights the importance of rigorous validation and cautious interpretation in chemometrics-based biomedical research to guarantee reliable biological and clinical conclusions.