Supplemental amino acids have improved muscle mass in horses and hoof structural formation in ruminants. Calcium supports sulfur crosslinks between hoof proteins, and fatty acids contribute to the periople, a barrier that sustains hoof horn quality. The objective was to test a hypothesis that a supplement containing essential amino acids lysine, threonine, methionine, fatty acids, and marine-based calcium would positively impact hoof growth and muscle mass in horses. A secondary objective was to develop a method for consistent measurement of hoof growth. The protocol was approved by the Institutional Animal Care and Use Committee. In a 24-wk crossover design, 12 horses aged 17 ± 7 yrs, 540 ± 65 kg BW, and 6.4 ± 0.8 BCS were fed prairie grass hay and 14% protein feed balanced to meet NRC requirements (CON), with or without 0.45 kg of added supplement (SUPPL), 6 horses on each diet per 12-wk period. Period 1 was Dec to Mar (winter), Period 2 Mar to Jun (spring). Horses were shod or trimmed throughout the trial and maintained in light to moderate work. To start, a cordless drill with 1.6 mm drill bit was used to mark the external horn of each front hoof, 20 mm below the coronary hairline and 2 mm in depth, deep enough to persist without any tissue damage. Every 2 wk, yellow Play-Doh was placed in the drill mark to improve visibility,and the distance between the coronary band and drill mark in each front hoof measured using a digital caliper (Penn Tool Co. Inc., Maplewood, NJ). Pictures of the caliper placement on each coronary band were taken on d 0 to provide consistent reference points for subsequent measurements. Photos for muscle mass scoring were taken at the end of each period, randomized, then scored post hoc by an observer blind to dietary treatment, with 1 = very little to 5 = significant muscle development, 3 = average. Data were analyzed using a mixed model with repeated measures (SAS Ver. 9.4). All horses consumed the supplement willingly with no feed refusals. The supplement did not impact hoof growth (16.7 ± 1.8 mm CON vs 15.9 ± 1.0 mm SUPPL; P = 0.21), BW (P = 0.76), or BCS (P = 0.64). Muscle mass scores were higher (P = 0.030) when horses were fed SUPPL (3.4 ± 0.2) vs CON (2.7 ± 0.2). Hoof growth was affected by season (P < 0.0001), greater in spring (20.3 ± 0.8 mm) vs winter (12.3 ± 0.8 mm). While 12-week provision of a supplement containing essential amino acids, fatty acids, and marine-based calcium did not impact hoof growth, muscle mass increased, and seasonal effects on hoof growth were noted.