A large body of evidence has linked unhealthy eating with an alarming increase in obesity and chronic disease worldwide. However, existing methods of assessing dietary intake rely on food frequency questionnaires or dietary records that are prone to bias and selective reporting. Herein, metabolic phenotyping was performed on 42 healthy participants from the Diet and Gene Intervention (DIGEST) pilot study, a parallel two-arm randomized clinical trial that provided complete diets to all participants. Matching urine and plasma specimens were collected at baseline and following 2 weeks of provision of either a Prudent or Western diet with a weight-maintaining menu plan designed by a dietician. Targeted and nontargeted metabolite profiling was conducted using three complementary analytical platforms, where 80 serum metabolites and 84 creatinine-normalized urinary metabolites were reliably measured (CV < 30%) in the majority of participants (> 75%) after implementing a rigorous data workflow for metabolite authentication with stringent quality control. We classified a panel of metabolites with distinctive trajectories following 2 weeks of food provisions when using complementary univariate and multivariate statistical models. Unknown metabolites associated with contrasting dietary patterns were identified with high resolution MS/MS and/or co-elution after spiking with authentic standards. Overall, 3-methylhistidine and proline betaine concentrations increased consistently when participants were assigned a Prudent diet (q < 0.05) in both plasma and urine samples with a decrease in the Western diet group. Similarly, creatinine-normalized urinary imidazole propionate, hydroxypipecolic acid, dihydroxybenzoic acid, and enterolactone glucuronide, as well as plasma ketoleucine and ketovaline increased with a Prudent diet (p < 0.05) after adjustments for age, sex and BMI. In contrast, plasma myristic acid, linoelaidic acid, linoleic acid, α-linoleic acid, pentadecanoic acid, alanine, proline, carnitine and deoxycarnitine, as well as urinary acesulfame K increased among participants following a Western diet. Most metabolites were also correlated (r > ± 0.30, p < 0.05) to changes in average intake of specific nutrients from self-reported diet records reflecting good adherence to food provisions. This study revealed robust biomarkers sensitive to short-term changes in habitual diet that can be used to reliably monitor healthy eating patterns for new advances in nutritional epidemiology, as well as the design of evidence-based public health policies for chronic disease prevention.